Systems and methods for detecting infectious diseases

ABSTRACT

Systems, methods, and devices for detecting infections in a clinical sample are provided. Small-volume clinical samples obtained at a point-of-service (POS) location and may be tested at the POS location for multiple markers for multiple diseases, including upper and lower respiratory diseases. Samples may be tested for cytokines, or for inflammation indicators. Dilution of samples, or levels of detection, may be determined by the condition or past history of a subject. Test results may be obtained within a short amount of time after sample placement in a testing device, or within a short amount of time after being obtained from the subject. A prescription for treatment of a detected disorder may be provided, and may be filled, at the POS location. A bill may be automatically generated for the testing, or for the prescription, may be automatically sent to an insurance provider, and payment may be automatically obtained.

BACKGROUND

Infectious diseases, whether or bacterial, viral, or other origin,present acute and chronic challenges to human health. Many commoninfections affect the respiratory tract. Respiratory tract diseases,particularly infectious respiratory diseases of viral and bacterialorigin, are prevalent in patients of all ages, although often are moreserious in the very young and the very old. Viruses include DNA virusesand RNA viruses. Bacteria include Gram positive and Gram negativebacteria, and may include mycoplasma (bacteria lacking cell walls). Inaddition to disease-causing bacteria, some diseases, such as, e.g.,respiratory diseases, may be caused by other microorganisms such asyeasts, fungi, and other small, disease-causing organisms.

An example of a common viral cause of respiratory (and other) disordersin patients is the influenza (“flu”) virus. Influenza (“flu”) refers todisease caused by one of several related RNA viruses of theOrthomyxoviridae family, typified by fever, headache, fatigue, and othersymptoms. There are different types of influenza; influenza A andinfluenza B are both about equally prevalent in humans. Identificationof the strain of flu in a sample can help suggest treatments, can helpsuggest preventive measures to be taken, and can help to track suchinfections in a population.

Examples of common bacterial causes of respiratory (and other) disordersin patients include whooping cough, pneumonia, and tuberculosis.Whooping cough is caused by Bordetella pertussis and is typified by fitsof violent coughing, which may persist for weeks. Pneumonia is the namegiven to respiratory disorders characterized by fluid in the lungs,coughing, fever, vomiting, fatigue, and other symptoms. Pneumonia may becaused by bacterial or viral infection; determination of the cause of aparticular case is critical in determining the course of treatment ofthe patient. Causes of pneumonia include Streptococcus pneumonia,Staphylococcus aureus, adenovirus, influenza viruses, respiratorysyncytial virus, Pneumocystis, jirovecii (a fungus), and other agents.Tuberculosis is caused by Mycobacterium tuberculosis, is typified bycough including spitting up blood, chest pain, chills, fever, nightsweats, and other symptoms, and may be fatal.

Agents that cause infectious respiratory diseases typically differbetween upper respiratory tract diseases and lower respiratory tractdisorders; thus, the variety or range of bacterial or viral agents foundin patients suffering from upper respiratory disorders may be differentthan those bacterial or viral agents found in patients suffering fromlower respiratory disorders. However, successful diagnosis and treatmentof respiratory diseases often requires identification of disease-causingorganisms present in a clinical sample obtained from a subjectsuffering, or suspected of suffering, from an infectious respiratorydisorder. Differentiating between organisms typical of upper respiratoryand those typical of lower respiratory disorders may also be critical inthe successful diagnosis and treatment of respiratory diseases. Inaddition, identification of other symptoms and sequelae of respiratorydisorders may aid the successful diagnosis and treatment of respiratorydiseases.

Sexually transmitted diseases, whether viral or bacterial, or otherwise,present particular public health problems since some patients arereluctant to acknowledge the risks of, or possible exposure to, suchdiseases, and may be reluctant to be tested for these diseases. However,lack of testing and resulting lack of information regarding diseasestatus may lead to increased spread of such diseases, and delaystreatment for those affected.

Some diseases may be detected by blood tests (e.g., dengue virus,Epstein-Barr virus, trypanosomal diseases, plasmodium diseases, andothers). Some diseases may be detected by analysis of swabs, or fluidobtained from swabs, such as throat swabs, nasal swabs, cheek swabs, orother swabs. Diseases may also be detected by analysis of urine samples,and other clinical samples.

In order to be effective in treating such infectious disorders, testingmust be timely. However, present methods and systems for testing areoften time-consuming, inconvenient for patients, may require samplecollection methods or amounts that are painful or uncomfortable forpatients, and may be expensive. Methods that require large amounts ofsample, or that require incubation of a sample for a day or days, areoften ineffective at timely detection or identification of the cause ofa respiratory disorder, and thus may not be helpful in the diagnosis ortreatment of infectious respiratory disorders.

In addition, many infectious respiratory disorders present many of thesame, or similar symptoms, so that useful and effective testing requirestesting for the presence of multiple agents, and of multiple kinds ofagents (e.g., viral, bacterial, and fungal). However, present methodsare often limited to testing for a single agent or kind of agent, oronly a small number of possible agents, limiting the utility of theresults and raising the likelihood that the causal agent may not beidentified.

Thus, improved methods, systems, and assays for the detection andidentification of agents that cause diseases, such as influenza,respiratory diseases, sexually transmitted diseases, blood diseases,viral diseases, bacterial diseases, and other diseases, are desired.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

Systems, methods, and devices for detecting the presence of markersindicative of one or more of a plurality of infectious agents in asingle clinical sample, or in a plurality of aliquots of a singleclinical sample, are provided. The systems, methods, and devicesdisclosed herein may be point-of-service systems, methods, and devices,configured for use at a point-of-service location, where apoint-of-service location may be a location at which a sample isobtained from a subject.

In embodiments, Applicants disclose systems, methods, and devices fortesting for presence of one or more of a plurality of markers indicativeof an infectious disease in a single small-volume clinical sample, oraliquots thereof. In embodiments, the system, method, or device is apoint-of service (POS) system, method or device. In embodiments, thesample is collected at the POS location, and is analyzed in a device atthe POS location. In embodiments, the analysis of the small-volumeclinical sample is completed in a short period of time. In embodiments,the infectious disease comprises a respiratory disease. In embodiments,the infectious disease comprises a respiratory disease selected from anupper respiratory disease and a lower respiratory disease. Inembodiments, the infectious disease comprises a sexually transmitteddisease.

In embodiments, Applicants disclose systems, methods, and devices fordetecting the presence of one or more of a plurality of markersindicative of an infectious disease in a single small-volume clinicalsample, or aliquots thereof. In embodiments, the system, method ordevice is a POS system, method or device. In embodiments, the sample iscollected at the POS location, and is analyzed in a device at the POSlocation. In embodiments, the analysis of the small-volume clinicalsample is completed in a short period of time.

In embodiments, the infectious disease is a bacterial disease, or aviral disease, or another type of disease, and the analysis of thesmall-volume clinical sample determines whether the infectious diseaseis a bacterial disease, a viral disease, or another type of disease. Thedetermination of the type of infectious disease aids in determining thetype of treatment to provide to the subject, e.g., where thedetermination indicates the infectious disease is a fungal disease, thesubject should be treated with anti-fungal drugs; where thedetermination indicates the infectious disease is a yeast infection, thesubject should be treated with anti-yeast drugs; and so forth.

In embodiments, the infectious disease is a bacterial disease. Inembodiments, the analysis of the small-volume clinical sample determineswhether the infectious disease is a bacterial disease. In embodimentswhere the analysis of the small-volume clinical sample determines thatthe infectious disease is a bacterial disease, said determinationindicates the use of antibiotics in the treatment of that disease. Inembodiments, the infectious disease is a viral disease. In embodiments,the analysis of the small-volume clinical sample determines whether theinfectious disease is a viral disease. In embodiments where the analysisof the small-volume clinical sample determines that the infectiousdisease is a viral disease, said determination indicates the use ofantiviral drugs in the treatment of that disease. In embodiments wherethe analysis of the small-volume clinical sample determines that theinfectious disease is a viral disease, said determination indicates thatantibiotics should not be used in the treatment of that disease. Inembodiments, the infectious disease is a bacterial disease, or a viraldisease. In embodiments, the analysis of the small-volume clinicalsample determines whether the infectious disease is a bacterial diseaseor a viral disease. Similarly, where the analysis of the small volumeclinical sample determines the infectious disease is a fungal disease,the subject should be treated with anti-fungal drugs; where thedetermination indicates the infectious disease is a yeast infection, thesubject should be treated with anti-yeast drugs; and so forth.

In embodiments, the infectious disease comprises a respiratory disease.In embodiments, the infectious disease comprises a respiratory diseaseselected from an upper respiratory disease and a lower respiratorydisease. In embodiments, the analysis of the small-volume clinicalsample determines whether the infectious disease is an upper respiratorydisease or a lower respiratory disease. In embodiments, the analysis ofthe small-volume clinical sample determines the type of upperrespiratory disease or a lower respiratory disease present in the smallvolume clinical sample. For example, in embodiments, the upper or lowerrespiratory disease is a bacterial disease, or a viral disease, oranother type of disease, and the analysis of the small-volume clinicalsample determines whether the upper or lower respiratory disease is abacterial disease, a viral disease, or another type of disease. Inembodiments where the analysis of the small-volume clinical sampledetermines that the upper or lower respiratory disease is a bacterialdisease, said determination indicates the use of antibiotics in thetreatment of that disease. In embodiments where the analysis of thesmall-volume clinical sample determines that the upper or lowerrespiratory disease is a viral disease, said determination indicates theuse of antiviral drugs in the treatment of that disease. In embodimentswhere the analysis of the small-volume clinical sample determines thatthe upper or lower respiratory disease is a viral disease, saiddetermination indicates that antibiotics should not be used in thetreatment of that disease. Similarly, where the analysis of the smallvolume clinical sample determines the upper or lower respiratory diseaseis a fungal disease, the subject should be treated with anti-fungaldrugs; where the determination indicates the infectious disease is ayeast infection, the subject should be treated with anti-yeast drugs;and so forth.

In embodiments, the infectious disease comprises a sexually transmitteddisease. In embodiments, the analysis of the small-volume clinicalsample determines the type of sexually transmitted disease present inthe small volume clinical sample. For example, in embodiments, thesexually transmitted disease is a bacterial disease, or a viral disease,or another type of disease, and the analysis of the small-volumeclinical sample determines whether the sexually transmitted disease is abacterial disease, a viral disease, or another type of disease. Inembodiments where the analysis of the small-volume clinical sampledetermines that the sexually transmitted disease is a bacterial disease,said determination indicates the use of antibiotics in the treatment ofthat disease. In embodiments where the analysis of the small-volumeclinical sample determines that the sexually transmitted disease is aviral disease, said determination indicates the use of antiviral drugsin the treatment of that disease. In embodiments where the analysis ofthe small-volume clinical sample determines that the sexuallytransmitted disease is a viral disease, said determination indicatesthat antibiotics should not be used in the treatment of that disease.Similarly, where the analysis of the small volume clinical sampledetermines the sexually transmitted disease is a fungal disease, thesubject should be treated with anti-fungal drugs; where thedetermination indicates the infectious disease is a yeast infection, thesubject should be treated with anti-yeast drugs; and so forth.

In embodiments of the systems, methods, and devices configured fortesting for a plurality of markers, and in systems, methods or devicesconfigured for detecting a plurality of markers, the markers may beindicative of respiratory diseases; in embodiments, the markers may beindicative of upper respiratory diseases; in embodiments, the markersmay be indicative of lower respiratory diseases. In embodiments,Applicants disclose systems, methods and devices configured for testingfor a plurality of markers, wherein the respiratory disease markers areindicative of two or more of the group of respiratory disease markersconsisting of adenovirus B, adenovirus C, adenovirus E, Bordetellapertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus,Methicillin-Resistant Staphylococcus aureus (MRSA), Group Astreptococcus, and Group B streptococcus. In embodiments, Applicantsdisclose systems, methods and devices configured for testing for aplurality of markers, wherein the respiratory disease markers areindicative of two or more of the group of respiratory disease markersconsisting of adenovirus B, adenovirus C, adenovirus E, Bordetellapertussis, Bordetella parapertussis, mycobacterium tuberculosis (MTB),Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus(MRSA), Group A streptococcus, Group B streptococcus, Moraxellacatarrhais, Enterobacter aerogenes, Haemophilus parainfluenzae,Metapneumo Virus, Streptococcus pneumonia, Parainfluenza Virus 1,Parainfluenza Virus 2, Parainfluenza Virus 3, Coronavirus OC43,Coronavirus NL63, Coronavirus MERS, Coronavirus HKU1, Coronavirus 229E,Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, Bocavirus type 2,4,and Bocavirus type 1,3. In embodiments, the respiratory disease markersare indicative of three or more, or of four or more, or of five or more,or of six or more, or of seven or more, or of eight of that group ofrespiratory disease markers.

In embodiments of the systems, methods, and devices configured fortesting for a plurality of markers, and in systems, methods, and devicesconfigured for detecting a plurality of markers, the markers may beindicative of sexually transmitted diseases. In embodiments, Applicantsdisclose systems, methods, and devices configured for testing for aplurality of markers, wherein the sexually transmitted disease markersare indicative of two or more of the group of markers consisting ofherpes simplex virus (HSV), human immunodeficiency virus (HIV),streptococcus B, and treponema pallidum. In embodiments, Applicantsdisclose systems, methods, and devices configured for testing for aplurality of markers, wherein the sexually transmitted disease markersare indicative of two or more of the group of markers consisting ofHIV-2 Group A, HIV-2, Group B, HIV-1 Group M, Hepatitis B, HepatitisDelta, herpes simplex virus (HSV), streptococcus B, and treponemapallidum. In embodiments, the sexually transmitted disease markers areindicative of three or more, or four of that group of sexuallytransmitted disease markers.

In embodiments of the systems, methods, and devices configured fortesting for a plurality of markers, and in systems, methods, and devicesconfigured for detecting a plurality of markers, the markers may beindicative of influenza. In embodiments, Applicants disclose systems,methods, and devices configured for testing for a plurality of markers,wherein the influenza markers are indicative of influenza A and ofinfluenza B. In embodiments, Applicants disclose systems, methods, anddevices configured for testing for a plurality of markers, wherein theinfluenza markers are indicative of two or more of the group of markersconsisting of the following forms of influenza: H1N1 (seasonal), H1N1(novel), H3N2, H7N9 (hemagglutinin gene marker (HA) and neuraminidasegene (NA)), and H5N1. In embodiments, the influenza markers areindicative of three or more, or four or more, or five of that group ofinfluenza markers. In embodiments, the influenza markers may beinfluenza Matrix Protein markers, or may be influenza neuraminidaseprotein markers, or may be influenza hemagglutinin markers, or otherinfluenza markers. In embodiments, the analysis of the small-volumeclinical sample determines whether the infectious disease is aninfluenza. In embodiments, the analysis of the small-volume clinicalsample determines the type of influenza present in the small volumeclinical sample. In embodiments where the analysis of the small-volumeclinical sample determines that the infectious disease is an influenza(which is a viral disease), said determination indicates thatantibiotics should not be used in the treatment of that disease. Inembodiments where the analysis of the small-volume clinical sampledetermines that the infectious disease is an influenza, saiddetermination indicates that antiviral drugs should be used in thetreatment of that disease.

In embodiments of the systems, methods, and devices configured fortesting for a plurality of markers, and in systems, methods, and devicesconfigured for detecting a plurality of markers, the markers may beindicative of diseases and disease markers which may be detected byanalysis of a blood sample. In embodiments, such diseases and diseasemarkers which may be detected by analysis of a blood sample include WestNile Virus, Epstein-Barr Virus, plasmodium, Trypanosoma cruzi, andDengue Virus (including types 1, 2, 3, and 4).

Samples from the throat of a subject may be obtained, e.g., by a throatswab; samples obtained from the nose of a subject may be obtained, e.g.,by a nasal swab. In embodiments, samples obtained from the throat andfrom the nose of a subject may be tested together. In embodiments,testing of samples obtained from the throat, or from the nose, or fromboth the nose and from the throat, may be tested by nucleic acidanalysis; or by amino acid analysis (e.g., ELISA or other antibody-basedor binding protein-based analysis); or by general chemistry analysis; orby cytometric analysis; or by combinations thereof. For example, samplesmay be tested by nucleic acid analysis and by amino acid analysis. Suchtests may be used to determine how long a subject has had an infection,for example, by noting the delay in rise of levels of antibodiesindicative of a particular disease in the sample; or by tracking therise in the levels of antibodies indicative of a particular disease inthe sample over time (e.g., by repeated testing over time). Similarly,such testing may be used to detect, or to determine, the effect oftreatment, by noting the delay in rise of levels of antibodiesindicative of a particular disease in the sample; or by tracking therise in the levels of antibodies indicative of a particular disease inthe sample over time (e.g., by repeated testing over time). Inembodiments, samples from throat and from nose may be included in asingle solution, and tested together. In embodiments, samples fromthroat and from nose may be in separate vessels (e.g., samplecontainers), but both included in a single cartridge, and the separatevessels tested at the same time. Such testing at the same time maycomprise testing the vessels separately, or may include mixing thecontents of the vessels and testing the mixture.

In embodiments, Applicants disclose systems, methods, and devicesconfigured for identifying, or estimating, or otherwise determining thestage of an infection in a subject by detecting, or determining theamounts of, or both, both nucleic acid markers indicative of aparticular infection and antibody markers indicative of the sameinfection. Such systems, methods, and devices may be used to detect,measure, and track such markers over time, effective to provide anestimate or determination of how recently an infection occurred. Suchsystems, methods, and devices may be used to detect, measure, and tracksuch markers over time, effective to aid in evaluating the presentstatus of a subject suffering from an infection. Such systems, methods,and devices may be used to detect, measure, and track such markers overtime, effective to aid in determining the likely prognosis of a subjectsuffering from an infection. For example, where nucleic acid markersindicative of a particular infection are relatively numerous, whileantibody or other protein markers indicative of that particularinfection are relatively sparse, then it can be estimated or determinedthat the infection is a recent infection; however, where nucleic acidmarkers indicative of a particular infection are relatively numerous,and antibody or other protein markers indicative of that particularinfection are also relatively numerous, then it can be estimated ordetermined that the infection is not a recent infection, since thesubject has had the time to produce infection-specific antibodies. Wherenucleic acid markers indicative of a particular infection are relativelysparse, and antibody or other protein markers indicative of thatparticular infection are also relatively numerous, then it can beestimated or determined that the infection in a late stage, andindicates that the infection is waning, since such observations indicatethat the subject is overcoming the infection.

In embodiments, Applicants disclose systems, methods, and devicesconfigured for testing for a plurality of markers, and disclose systemsconfigured for detecting a plurality of markers, where the markers areindicative of a plurality of infectious diseases in a singlesmall-volume clinical sample, or aliquots thereof. In embodiments, thesystems, methods, and devices may be configured for testing for, or fordetecting, markers indicative of more than about 8 different diseases,or more than about 8 different diseases, or more than about 12 differentdiseases, or more than about 16 different diseases, or more than about20 different diseases, or more than about 25 different diseases, or morethan about 35 different diseases, or more than about 45 differentdiseases, or more than about 60 different diseases. In embodiments, thesystems, methods, and devices may be configured to test for, or todetect a plurality of nucleic acid markers and protein markers, eachmarker being indicative of at least one of a plurality of diseases orconditions. In embodiments, the systems, methods, and devices may beconfigured to test for, or to detect a plurality of nucleic acidmarkers, protein markers, and cytometric markers, each marker beingindicative of at least one of a plurality of diseases or conditions. Inembodiments, the systems, methods, and devices may be configured to testfor, or to detect a plurality of nucleic acid markers, protein markers,cytometric markers, cytokines, and markers of inflammation, each markeror cytokine being indicative of at least one of a plurality of diseasesor conditions. In embodiments, the systems, methods, and devicescomprise a point-of service system. In embodiments, the sample may becollected at the point of service, and may be analyzed in a device atthe POS location. In embodiments, the analysis of the small-volumeclinical sample may be completed in a short period of time.

In embodiments, the infectious disease comprises a respiratory disease.In embodiments, the infectious disease comprises a respiratory diseaseselected from an upper respiratory disease and a lower respiratorydisease. In embodiments, the analysis of the small-volume clinicalsample determines whether the infectious disease is an upper respiratorydisease or a lower respiratory disease. In embodiments, the analysis ofthe small-volume clinical sample determines the type of upperrespiratory disease or a lower respiratory disease present in the smallvolume clinical sample. In embodiments, the respiratory diseasecomprises a respiratory disease caused by a disease-causing agentselected from a virus, a bacterium, a yeast, a fungus, a mycoplasma, andother micro-organisms. In embodiments, the analysis of the small-volumeclinical sample determines the type of upper respiratory disease or alower respiratory disease present in the small volume clinical sample.In embodiments, the analysis of the small-volume clinical sampledetermines whether the upper respiratory disease or a lower respiratorydisease is a viral disease, or a bacterial disease, or some other typeof disease. In embodiments in which the analysis of the small-volumeclinical sample determines that the disease is a viral disease, saiddetermination indicates that antibiotics should not be used in thetreatment of that disease. In embodiments where the analysis of thesmall-volume clinical sample determines that the disease is a viraldisease, said determination indicates that antiviral drugs should beused in the treatment of that disease. In embodiments in which theanalysis of the small-volume clinical sample determines that the diseaseis a bacterial disease, said determination indicates that antibioticsshould be used in the treatment of that disease.

In embodiments, the infectious disease comprises a sexually transmitteddisease. In embodiments, the sexually transmitted disease comprises asexually transmitted disease caused by a disease-causing agent selectedfrom a virus, a bacterium, a yeast, a fungus, a mycoplasma, and othermicro-organisms. In embodiments, the analysis of the small-volumeclinical sample determines the type of sexually transmitted diseasepresent in the small volume clinical sample. In embodiments, theanalysis of the small-volume clinical sample determines whether thesexually transmitted disease is a viral disease, or a bacterial disease,or some other type of disease. In embodiments in which the analysis ofthe small-volume clinical sample determines that the disease is a viraldisease, said determination indicates that antibiotics should not beused in the treatment of that disease. In embodiments where the analysisof the small-volume clinical sample determines that the disease is aviral disease, said determination indicates that antiviral drugs shouldbe used in the treatment of that disease. In embodiments in which theanalysis of the small-volume clinical sample determines that the diseaseis a bacterial disease, said determination indicates that antibioticsshould be used in the treatment of that disease.

Applicant further discloses a method of determining the state ofresponse to a disease in a subject, the method comprising: a)introducing a clinical sample into a sample processing device, saidsample having been obtained from a subject suspected of suffering from adisease caused by a disease-causing organism, said clinical samplehaving a volume of no greater than 500 microliters, wherein the devicecomprises: i) a sample handling system; ii) a detection station; andiii) an assay station comprising at least a first and a secondindependently movable assay unit; b) with the aid of the sample handlingsystem, transferring a portion of the clinical sample to each of thefirst and second assay units, wherein an assay for the detection of anucleic acid indicative of the disease-causing organism is performed insaid first assay unit, and an assay for the detection of antibodies tothe disease-causing organism is performed in the second assay unit; c)transferring the first and second assay units to the detection stationwith the aid of the sample handling system; d) obtaining datameasurements with the aid of the detection station, said datameasurements comprising determining the level of nucleic acid indicativeof a disease organism in the sample, and determining the level ofantibodies directed to that disease organism in the sample; and e) i)determining that the infection is a recent infection, and in an earlystage of the disease, where the level of nucleic acid indicative of adisease organism is high and the level of antibodies directed to thatdisease organism is low or normal; ii) determining that the infection isnot a recent infection, and not in an early stage of the disease, wherethe level of nucleic acid indicative of a disease organism is high andthe level of antibodies directed to that disease organism is high; andiii) determining that the infection is a waning infection, and in a latestage of the disease, where the level of nucleic acid indicative of adisease organism is low or normal and the level of antibodies directedto that disease organism is high, where a normal level of a marker isthe level of that marker determined in a healthy population of normalsubjects, where a high level is one that significantly exceeds a normallevel as determined in a healthy population, and a low level is one thatis at or below the normal level as determined in a healthy population.

In embodiments, the method of determining the state of response to adisease in a subject further comprises detecting the level ofinflammatory cytokines. In embodiments of the method of determining thestate of response to a disease in a subject, the device furthercomprises a cytometry station comprises an imaging device and a stagefor receiving a microscopy cuvette, and the method further comprisesimaging a white blood cell in a blood sample obtained from the subject.In embodiments, imaging a white blood cell in a blood sample obtainedfrom the subject comprises detecting the level of a white blood celltype in a blood sample obtained from the subject, and determiningwhether said detected level of said type of white blood cell is above,at, or below a normal level for that type of blood cell, wherein thenormal level for that type of white blood cell is determined by thelevel of that type of white blood cell in blood samples from a healthypopulation.

Tests may be for the detection of markers indicative of any infectiousdisease. For example, diseases that may be tested for includerespiratory diseases, and include upper respiratory diseases and lowerrespiratory diseases. Markers may include nucleic acid markers, proteinmarkers, polysaccharide markers, cellular markers (including cells andcellular organelles or fragments), and other markers. Markers mayinclude markers for viral infections, bacterial infections, fungalinfections, yeast infections, mycoplasma infections, and for otherinfections. Samples may be tested for markers indicative ofinflammation. Samples may be tested for cytokines. Samples may be testedfor inflammatory cytokines. Samples may be tested for anti-inflammatorycytokines. The amount of dilution of a sample, or a level of detectionof a marker, may be determined by the condition or past history of asubject.

Test results may be obtained within three hours, or two hours, or onehour, or ½ hour, or less from the time a sample is placed in a testingdevice for analysis. A sample may be placed in a testing device foranalysis within five hours, or four hours, or three hours, or two hours,or one hour, or ½ hour, or less from the time a sample was obtained froma subject. Test results may be obtained within eight hours, seven hours,or six hours, or five hours, or three hours, or two hours, or one hour,or ½ hour, or less from the time a sample was obtained from a subject.

In embodiments, Applicant discloses a method of detecting a diseasemarker, comprising: a) introducing a cartridge comprising one or moresamples into an automatic sample processing device, said cartridge beingconfigured to hold at least one sample and being configured to hold aswab, wherein said automatic sample processing device comprises: i) asample handling system configured to transport at least a portion of asample and being configured to transport an independently movable assayunit; and ii) an optical detector; b) contacting a sample, or a portionthereof, with a movable assay unit, or a reagent, or both, for theperformance of an assay for the detection of a disease marker, saidcontacting comprising transporting, with the aid of said sample handlingsystem, at least a portion of said sample, or a movable assay unit, or areagent, or combinations thereof; c) positioning said sample, or portionthereof, at a location suitable for detection of an optical signal fromthe sample or portion thereof by said optical detector; and d) detectingthe presence of a disease marker. In embodiments, such a method maycomprise performing two or more assays for the detection of diseasemarkers, and detecting two or more disease markers in said one or moresamples, or in one or more portions thereof. In embodiments, the samplehas a volume of less than about 500 microliters (L). In embodiments, theone or more samples comprises a blood sample; or comprises a sampleobtained using a swab; or comprises both a blood sample and a sampleobtained using a swab. In embodiments, a sample obtained using said swabmay be obtained by swabbing a mouth, a throat, a nasal passage, avaginal area, or other body cavity of a subject. In embodiments, themethod comprises detecting the presence of a nucleic acid disease markerand a protein disease marker.

The methods disclosed herein include performing two or more assays forthe detection of disease markers, and detecting two or more diseasemarkers in said samples, or in one or more portions thereof. Inembodiments, the methods comprise detecting the presence of a nucleicacid disease marker and a protein disease marker. In embodiments, thedisease marker is selected from a nucleic acid disease marker, a proteindisease marker, a saccharide, a prostaglandin, a cytokine, histamine, asteroid, and a marker for inflammation. In embodiments, two or moredisease markers are detected, wherein the disease markers are selectedfrom a nucleic acid disease marker, a protein disease marker, asaccharide, a prostaglandin, a cytokine, histamine, a steroid, and amarker for inflammation. In embodiments, a disease marker is a markerfor inflammation selected from prostaglandins, tumor necrosis factoralpha (TNF-α), interleukin-1 (IL-1), interleukin-8 (IL-8),interleukin-12 (IL-12), interferon gamma (IF-γ), bradykinin, complementsystem molecules, blood-clotting factors, C-reactive protein,erythrocyte sedimentation rate (ESR), white blood cell count, andmorphological changes in blood and other cells.

In embodiments, a disease marker is a marker for a disease-causingagent, wherein said disease-causing agent is selected from the group ofdisease-causing organisms consisting of a virus, a bacterium, amycoplasm, a fungus, a yeast, and other micro-organisms. In embodiments,a disease marker for a disease-causing agent is selected from the groupconsisting of Influenza A Matrix protein, Influenza H3N2, Influenza H1N1seasonal, Influenza H1N1 novel, Influenza B, Streptococcus pyogenes (A),Mycobacterium Tuberculosis, Staphylococcus aureus (MR), Staphylococcusaureus (RS), Bordetella pertussis (whooping cough), Streptococcusagalactiae (B), Influenza H5N1, Influenza H7N9, Adenovirus B, AdenovirusC, Adenovirus E, Hepatitis b, Hepatitis c, Hepatitis delta, Treponemapallidum, HSV-1, HSV-2, HIV-1, HIV-2, Dengue 1, Dengue 2, Dengue 3,Dengue 4, Malaria, West Nile Virus, Trypanosoma cruzi (Chagas),Klebsiella pneumoniae (Enterobacteriaceae spp), Klebsiella pneumoniaecarbapenemase (KPC), Epstein Barr Virus (mono), Rhinovirus,Parainfluenza virus (1), Parainfluenza virus (2), Parainfluenza virus(3), Parainfluenza virus (4a), Parainfluenza virus (4b), Respiratorysyncytial virus (RSV) A, Respiratory syncytial virus (RSV) B,Coronavirus 229E, Coronavirus HKU1, Coronavirus OC43, Coronavirus NL63,Novel Coronavirus, Bocavirus, human metapneumovirus (HMPV),Streptococcus pneumoniae (penic R), Streptococcus pneumoniae (S),Mycoplasma pneumoniae, Chlamydia pneumoniae, Bordetella parpertussis,Haemophilus influenzae (ampic R), Haemophilus influenzae (ampic S),Moraxella catarrhalis, Pseudomonas spp (aeruginosa), Haemophilusparainfluenzae, Enterobacter cloacae (Enterobacteriaceae spp),Enterobacter aerogenes (Enterobacteriaceae spp), Serratia marcescens(Enterobacteriaceae spp), Acinetobacter baumanii, Legionella spp,Escherichia coli, Candida, Chlamydia trachomatis, Human Papilloma Virus,Neisseria gonorrhoeae, plasmodium, and Trichomonas (vagin).

In embodiments, the methods comprise detecting a disease marker in ablood sample and detecting a disease marker in a sample obtained from aswab, wherein one of said disease markers is a marker for inflammation,and one of said disease markers a marker for a disease-causing agent. Inembodiments, such a disease marker for inflammation is selected fromprostaglandins, tumor necrosis factor alpha (TNF-α), interleukin-1(IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interferon gamma(IF-γ), bradykinin, complement system molecules, blood-clotting factors,C-reactive protein, erythrocyte sedimentation rate (ESR), white bloodcell count, and morphological changes in blood and other cells, and sucha disease marker for a disease-causing agent is selected from the groupof disease-causing organisms consisting of a virus, a bacterium, amycoplasm, a fungus, a yeast, and other micro-organisms.

In embodiments, a disease marker is a marker for a disease selected frominfluenza, a respiratory disease, a sexually transmitted disease, andanother infectious disease. In embodiments where the disease isinfluenza, the disease marker is selected from H1N1 (seasonal), H1N1(novel), H3N2, H7N9, and H5N1. In embodiments, the disease isrespiratory disease selected from an upper respiratory disease and alower respiratory disease. In embodiments where the disease is arespiratory disease, the marker may be a marker for a disease-causingorganism selected from the group consisting of adenovirus B, adenovirusC, adenovirus E, Bordetella pertussis, mycobacterium tuberculosis (MTB),Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus(MRSA), Group A streptococcus, Group B streptococcus, Moraxellacatarrhalis, Enterobacter aerogenes, Haemophilus parainfluenzae,Metapneumo Virus, Streptococcus pneumonia, Parainfluenza Virus 1,Parainfluenza Virus 2, Parainfluenza Virus 3, Coronavirus OC43,Coronavirus NL63, Coronavirus MERS, Coronavirus HKU1, Coronavirus 229E,Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, Bocavirus type 2,4,and Bocavirus type 1,3.

In embodiments where the disease is a sexually transmitted disease, themarker may comprise a marker for a disease-causing organism indicativeof a sexually transmitted disease selected from herpes simplex virus(HSV), human immunodeficiency virus (HIV), HIV-2 Group A, HIV-2 Group B,HIV-1 Group M, Hepatitis B, Hepatitis Delta, herpes simplex virus (HSV),streptococcus B, and treponema pallidum.

In embodiments, the method comprises detecting a disease marker in ablood sample and detecting a disease marker in a sample obtained from aswab, wherein at least one of said disease markers is a marker for adisease-causing organism indicative of a respiratory disease selectedfrom the group consisting of adenovirus B, adenovirus C, adenovirus E,Bordetella pertussis, mycobacterium tuberculosis (MTB), Staphylococcusaureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Group Astreptococcus, Group B streptococcus, Moraxella catarrhalis,Enterobacter aerogenes, Haemophilus parainfluenzae, Metapneumo Virus,Streptococcus pneumonia, Parainfluenza Virus 1, Parainfluenza Virus 2,Parainfluenza Virus 3, Coronavirus OC43, Coronavirus NL63, CoronavirusMERS, Coronavirus HKU1, Coronavirus 229E, Klibsiella pneumonia phoE,Klebsiella pneumonia KPC, Bocavirus type 2,4, and Bocavirus type 1,3.

In embodiments where the disease is an infectious disease, the diseasemarker may comprise a marker for an infectious disease-causing agentselected from the group consisting of West Nile Virus, Epstein-BarrVirus, plasmodium, Trypanosoma cruzi, and a Dengue Virus.

In embodiments comprising detecting a disease marker in a blood sampleand detecting a disease marker in a sample obtained from a swab, whereinat least one of said disease markers is a marker for a disease-causingorganism indicative of a sexually transmitted disease selected fromherpes simplex virus (HSV), human immunodeficiency virus (HIV), HIV-2Group A, HIV-2 Group B, HIV-1 Group M, Hepatitis B, Hepatitis Delta,herpes simplex virus (HSV), streptococcus B, and treponema pallidum.

In embodiments comprising detecting a disease marker in a blood sampleand detecting a disease marker in a sample obtained from a swab, whereinat least one of said disease markers is a marker for a disease-causingagent selected from the group consisting of West Nile Virus,Epstein-Barr Virus, plasmodium, Trypanosoma cruzi, and a Dengue Virus.

In embodiments of the methods of detecting a disease marker, the methodis a point-of service method performed at a point-of-service location.In embodiments of methods of detecting a disease marker, the method maybe performed in less than about 40 minutes. In embodiments comprisingdetecting a disease marker in a blood sample and detecting a diseasemarker in a sample obtained from a swab, the method is a point-ofservice method performed at a point-of-service location. In embodimentscomprising detecting a disease marker in a blood sample and detecting adisease marker in a sample obtained from a swab, the method may beperformed in less than about 40 minutes.

For example, Applicant discloses herein a method of determining thestage of an infection in a subject suffering from an infection,comprising: Testing at least one sample, or an aliquot or aliquotsthereof, obtained from said subject 1) for the presence of a nucleicacid indicative of the infection, and 2) for the presence of an antibodyindicative of the infection, and Determining whether the relativeamounts of a) nucleic acids indicative of the infection and b)antibodies indicative of the infection indicate that the infection is arecent infection, wherein i) a greater relative amount of the nucleicacids indicative of the infection as compared to the relative amount ofthe antibodies indicative of the infection indicate that the infectionis a recent infection, and ii) a significant amount of antibodies to theinfectious agent indicate the infection is not a recent infection. Inembodiments of such a method, the at least one sample comprises a bloodsample. In embodiments of such a method, the at least one samplecomprises a sample selected from a throat swab sample, a cheek swabsample, nasal swab sample, a saliva sample, and a blood sample. Inembodiments of such a method, where a significant amount of antibody tothe infectious agent is detected, and where nucleic acid markersindicative of the infectious agent are relatively sparse, then themethod indicates that the infection is in a late stage and that theinfection is waning.

In embodiments, such a method may further include testing a sample orsamples for a marker for inflammation. In embodiments, the marker forinflammation may be selected from a prostaglandin, tumor necrosis factoralpha (TNF-α), interleukin-1 (IL-1), interleukin-8 (IL-8),interleukin-12 (IL-12), interferon gamma (IF-γ), bradykinin, acomplement system molecule, a blood-clotting factor, and a morphologicalchange in a blood or other cell. In embodiments, the marker forinflammation is a cytokine selected from a lymphokine, a chemokine, aninterleukin, and an interferon.

In embodiments, a method disclosed herein comprises testing to determinewhether the subject suffers from a bacterial infection, a viralinfection, a yeast infection, a mycoplasma infection, a fungalinfection, other infection, or combination thereof. In embodiments, suchtesting comprises determining whether markers indicative of viralinfection or markers indicative of bacterial infection are detected,effective to determine whether the subject suffers from a bacterialinfection or a viral infection.

In embodiments of the methods disclosed herein, the methods furthercomprise prescribing a prescription suitable for treatment of theinfection. In embodiments, methods disclosed herein comprise providing aprescription suitable for treatment of said infection comprisesprescription of an antibiotic when the testing determines that thesubject suffers from a bacterial infection. In embodiments, methodsdisclosed herein comprise providing a prescription suitable fortreatment of said infection comprises the prescription of ananti-mycoplasmal drug when the testing determines that the subjectsuffers from a mycoplasmal infection. In embodiments, methods disclosedherein comprise providing a prescription suitable for treatment of saidinfection comprises the prescription of an anti-viral drug when thetesting determines that the subject suffers from a viral infection. Inembodiments, methods disclosed herein comprise providing a prescriptionsuitable for treatment of said infection comprises avoiding theprescription of an antibiotic, when the testing determines that thesubject suffers from a viral infection. In embodiments, methodsdisclosed herein comprise providing a prescription suitable fortreatment of said infection comprises avoiding the prescription of anantibiotic, and providing the prescription of an anti-viral drug, whenthe testing determines that the subject suffers from a viral infection.In embodiments, methods disclosed herein comprise providing aprescription suitable for treatment of said infection comprises theprescription of an anti-fungal drug when the testing determines that thesubject suffers from a fungal infection. In embodiments, methodsdisclosed herein comprise providing a prescription suitable fortreatment of said infection comprises the prescription of an anti-yeastdrug when the testing determines that the subject suffers from a yeastinfection.

In embodiments, the methods comprise detecting a disease, wherein thedisease detected is caused by a disease-causing agent selected from thegroup of disease-causing organisms consisting of a virus, a bacterium, amycoplasm, a fungus, a yeast, and other micro-organisms, and furthercomprising providing a prescription for the suitable treatment of saidvirus, bacterium, mycoplasm, fungus, yeast, or other micro-organism.

In embodiments, the methods are point-of service methods performed at apoint-of-service location. In embodiments, the methods compriseperforming a plurality of assays on a single small-volume clinicalsample, or on aliquots thereof, and may be performed in less than about40 minutes. In embodiments, the infection is caused by a diseaseselected from influenza, a respiratory disease, a sexually transmitteddisease, and another infectious disease. In embodiments where theinfection comprises influenza, the influenza may be selected from H1N1(seasonal), H1N1 (novel), H3N2, H7N9, and H5N1. In embodiments where theinfection comprises a respiratory disease, the infection may be selectedfrom an upper respiratory disease and a lower respiratory disease. Inembodiments, the respiratory disease is selected from the groupconsisting of adenovirus B, adenovirus C, adenovirus E, Bordetellapertussis, mycobacterium tuberculosis (MTB), Staphylococcus aureus,Methicillin-Resistant Staphylococcus aureus (MRSA), Group Astreptococcus, Group B streptococcus, Moraxella catarrhalis,Enterobacter aerogenes, Haemophilus parainfluenzae, Metapneumo Virus,Streptococcus pneumonia, Parainfluenza Virus 1, Parainfluenza Virus 2,Parainfluenza Virus 3, Coronavirus OC43, Coronavirus NL63, CoronavirusMERS, Coronavirus HKU1, Coronavirus 229E, Klibsiella pneumonia phoE,Klebsiella pneumonia KPC, Bocavirus type 2,4, and Bocavirus type 1,3.

In embodiments, the infection comprises a sexually transmitted diseaseselected from a disease caused by herpes simplex virus (HSV), humanimmunodeficiency virus (HIV), HIV-2 Group A, HIV-2 Group B, HIV-1 GroupM, Hepatitis B, Hepatitis Delta, herpes simplex virus (HSV),streptococcus B, and treponema pallidum. In embodiments, the infectioncomprises a disease caused by an infectious disease-causing agentselected from the group consisting of Influenza A Matrix protein,Influenza H3N2, Influenza H1N1 seasonal, Influenza H1N1 novel, InfluenzaB, Streptococcus pyogenes (A), Mycobacterium Tuberculosis,Staphylococcus aureus (MR), Staphylococcus aureus (RS), Bordetellapertussis (whooping cough), Streptococcus agalactiae (B), InfluenzaH5N1, Influenza H7N9, Adenovirus B, Adenovirus C, Adenovirus E,Hepatitis b, Hepatitis c, Hepatitis delta, Treponema pallidum, HSV-1,HSV-2, HIV-1, HIV-2, Dengue 1, Dengue 2, Dengue 3, Dengue 4, Malaria,West Nile Virus, Trypanosoma cruzi (Chagas), Klebsiella pneumoniae(Enterobacteriaceae spp), Klebsiella pneumoniae carbapenemase (KPC),Epstein Barr Virus (mono), Rhinovirus, Parainfluenza virus (1),Parainfluenza virus (2), Parainfluenza virus (3), Parainfluenza virus(4a), Parainfluenza virus (4b), Respiratory syncytial virus (RSV) A,Respiratory syncytial virus (RSV) B, Coronavirus 229E, Coronavirus HKU1,Coronavirus OC43, Coronavirus NL63, Novel Coronavirus, Bocavirus, humanmetapneumovirus (HMPV), Streptococcus pneumoniae (penic R),Streptococcus pneumoniae (S), Mycoplasma pneumoniae, Chlamydiapneumoniae, Bordetella parpertussis, Haemophilus influenzae (ampic R),Haemophilus influenzae (ampic S), Moraxella catarrhalis, Pseudomonas spp(aeruginosa), Haemophilus parainfluenzae, Enterobacter cloacae(Enterobacteriaceae spp), Enterobacter aerogenes (Enterobacteriaceaespp), Serratia marcescens (Enterobacteriaceae spp), Acinetobacterbaumanii, Legionella spp, Escherichia coli, Candida, Chlamydiatrachomatis, Human Papilloma Virus, Neisseria gonorrhoeae, plasmodium,and Trichomonas (vagin).

In embodiments, the infection comprises a bacterial infection caused bybacteria selected from the group consisting of Bordetella pertussis,mycobacterium tuberculosis (MTB), Staphylococcus aureus,Methicillin-Resistant Staphylococcus aureus (MRSA), Group Astreptococcus, Group B streptococcus, Moraxella catarrhalis,Enterobacter aerogenes, Haemophilus parainfluenzae, Streptococcuspneumonia, Klibsiella pneumonia phoE, Klebsiella pneumonia KPC, andtreponema pallidum.

In embodiments, the infection comprises a viral infection caused by avirus selected from the group consisting of an influenza virus, herpessimplex virus (HSV), human immunodeficiency virus (HIV), HIV-2 Group A,HIV-2 Group B, HIV-1 Group M, Hepatitis B, Hepatitis Delta, herpessimplex virus (HSV), West Nile Virus, Epstein-Barr Virus, a DengueVirus, adenovirus B, adenovirus C, adenovirus E, Metapneumo Virus,Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3,Coronavirus OC43, Coronavirus NL63, Coronavirus MERS, Coronavirus HKU1,Coronavirus 229E, Bocavirus type 2,4, and Bocavirus type 1,3. Inembodiments of influenza infections, the influenza may be selected fromH1N1 (seasonal), H1N1 (novel), H3N2, H7N9, and H5N1 influenza viruses.In embodiments, a viral infection comprises infection by a Dengue virus,wherein said Dengue virus is selected from Dengue virus type 1, Denguevirus type 2, Dengue virus type 3, and Dengue virus type 4.

A bill for the testing may be automatically generated at the POS. Theamount of the bill may be calculated per the tests performed, orpursuant to the results of the testing. A bill for the testing may beautomatically sent to the subject's insurance provider. Payment for thetesting may be automatically obtained from the subject, or from thesubject's insurance carrier, or from another source.

A prescription for treatment of a detected disorder may be provided atthe POS location. A prescription for treatment of a detected disordermay be filled at the POS location. A bill for the filled prescriptionmay be automatically generated. A bill for the prescription may beautomatically sent to the subject's insurance provider. Payment for thefilled prescription may be automatically obtained from the subject, orfrom the subject's insurance carrier, or from another source.

Accordingly, Applicants provide systems, methods, and devices for rapidanalysis of a small-volume clinical sample in a short period of time.Such rapid analysis includes testing for the presence of markersindicative of a plurality of disease-causing agents in a short period oftime. In embodiments, such disease-causing agents include agents whichcause upper respiratory disorders, and include agents which cause lowerrespiratory disorders. In embodiments, such systems, methods, anddevices are configured to detect one or more indicators of inflammation.In embodiments, such systems, methods, and devices are configured todetect one or more cytokines. In embodiments, such systems, methods, anddevices are configured to detect one or more inflammatory cytokines. Inembodiments, such systems, methods, and devices are configured to detectone or more anti-inflammatory cytokines.

In embodiments, Applicants provide systems, methods, and devices fordetecting a plurality of disease-causing agents in a single clinicalsample, or in a plurality of aliquots of a single clinical sample. Inembodiments, a single clinical sample may be a small volume clinicalsample of blood, sputum, tears, nasal swabs, throat swabs, mouth swabs(e.g., cheek swabs), vaginal swabs, or other bodily fluid, tissue,secretion, or excretion taken from a subject. In embodiments, a singleclinical sample has a volume of less than about 500 μL, or less thanabout 250 μL, or less than 150 μL, or less than about 100 μL, or lessthan about 50 μL, or less than about 25 μL, or less than about 10 μL, orless than about 5 μL, or less than about 1 μL, or less.

In embodiments, clinical samples may be obtained at a point-of-service(POS) location. A POS location may be, for example, a retail pharmacy, asupermarket, a hospital, a clinic, a physician's office, or otherlocation. Clinical samples may be tested at the POS location formultiple markers indicative of agents which may cause one or more of aplurality of diseases (e.g., at least 8, or at least 10, or at least 12,or at least 20, or at least 30, or at least 40, or at least 50, or atleast 60, or more markers, indicative of the same or similar numbers ofdifferent diseases). The testing may be completed in a short period oftime. In embodiments, the short period of time may be measured from thetime the sample is inserted into a device or system for performing ananalysis. In embodiments, the short period of time may be measured fromthe time the sample is obtained from the subject.

In embodiments, clinical samples may be analyzed at a POS location. Inembodiments, clinical samples obtained at a POS location may be analyzedat the same POS location. In embodiments, clinical samples may beobtained at a point-of-service (POS) location and may be analyzed at adifferent location. In embodiments, clinical samples may be analyzed ina short period of time, e.g., in a period of time that is less thanabout 5 hours, or less than about 4 hours, or less than about 3 hours,or less than about 2 hours, or less than about 1 hour, or less thanabout half an hour.

In embodiments, Applicants provide devices (e.g., cartridges) for use inperforming assays for detecting a plurality of disease-causing agents ina single clinical sample, or in a plurality of aliquots of a singleclinical sample. In embodiments, such a device may comprise a pluralityof vessels containing reagents for use in an assay for the detection ofa plurality of markers indicative of an infectious agent, e.g., an upperrespiratory infectious agent; a lower respiratory infectious agent; asexually transmitted disease-causing agent; an agent detectable from asample obtained from a swab (e.g., a throat swab, a nasal swab, or otherswab); an agent detectable from a blood sample; or combinations thereof.In embodiments, a device may be a cartridge configured to contain aplurality of reagent vessels. In embodiments, a device may be acartridge configured to contain a reagent vessel containing a reagentfor detecting a marker indicative of a disease-causing agent. Inembodiments, a device may be a cartridge configured to contain aplurality of reagent vessels containing reagents for detecting a markerindicative of a disease-causing agent. In embodiments, such adisease-causing agent, or such a plurality of disease-causing agents,includes disease-causing agents which cause upper respiratory disorders.In embodiments, such a disease-causing agent, or such a plurality ofdisease-causing agents, includes disease-causing agents which causelower respiratory disorders. In embodiments, such a disease-causingagent, or such a plurality of disease-causing agents, includesdisease-causing agents which cause sexually transmitted diseases. Inembodiments, such a disease-causing agent, or such a plurality ofdisease-causing agents, includes disease-causing agents which may bedetected in a blood sample. In embodiments, such a disease-causingagent, or such a plurality of disease-causing agents, includesdisease-causing agents which may be detected in a sample obtained with aswab, such as a throat swab, or a nasal swab, or a cheek swab, or othersample, or combinations thereof.

In embodiments, devices for use in performing assays for detecting aplurality of disease-causing agents as disclosed herein may furtherinclude a space, or a vessel, for holding a swab; or may further includea space, or a vessel, for holding two swabs, or a plurality of swabs. Inembodiments, such devices may further include two spaces, or twovessels, for holding two swabs; or may include a plurality of spaces, orof vessels, for holding a plurality of swabs. In embodiments, a singleswab may be placed in a single space, or vessel; in embodiments, twoswabs may be placed in a single space, or vessel; and in embodiments, aplurality of swabs may be placed in a single space, or vessel. Thus, inembodiments, a swab may be placed in a vessel, and, in embodiments, aswab, or a plurality of swabs, may be placed in a single vessel. Inembodiments, a plurality of swabs may be placed in a plurality ofvessels. Such a vessel for holding a swab, or such vessels for holdingswabs, may contain a reagent, or a diluent, or other solution for usewith a swab or swabs. In embodiments, a vessel for holding a swab may beused to provide a clean swab for use in obtaining a sample. Inembodiments, a vessel for holding a swab may be used to i) provide aclean swab for use in obtaining a sample and also to ii) receive theswab after its use in obtaining a sample from a subject. In embodiments,a vessel for holding a plurality of swabs may be used to provide aplurality of clean swabs for use in obtaining a sample. In embodiments,a vessel for holding a plurality of swabs may be used to i) provide aplurality of clean swabs for use in obtaining a sample and also to ii)receive one or more of the plurality of swabs after its use in obtaininga sample from a subject.

For example, a throat swab and a nasal swab may be obtained from asubject. A nasal swab may be useful for testing for upper respiratorydiseases, and a throat swab may be useful for testing lower respiratorydiseases. In embodiments, the throat swab may be placed in one vessel ina device (e.g., a cartridge), and the nasal swab may be placed in adifferent vessel in the device. These vessels may contain a reagent, ora diluent, or other solution for use with the swabs; such reagents maybe different for the throat swab and the nasal swab. In embodiments, thethroat swab and the nasal may be placed in the same vessel in a device.The vessel may contain a reagent, or a diluent, or other solution foruse with these swabs. The device may be placed in an analysis device, orwithin an analysis system, for analysis. Such analysis devices andanalysis systems may be placed at the same location as that where thesample was obtained; or such analysis devices and analysis systems maybe at a different location or locations than the location where thesample was obtained.

In embodiments, a device may be or comprise a cartridge configured tocontain a reaction vessel or a plurality of reagent vessels. Inembodiments, a device may be or comprise a cartridge configured tocontain a reaction vessel or a plurality of reaction vessels. Inembodiments, a device may be a cartridge configured to contain acytometry cuvette, or a plurality of cytometry cuvettes. In embodiments,a device may be or comprise a cartridge configured to contain a wastecontainer, or a plurality of waste containers. In embodiments, a devicemay be or comprise a cartridge configured to contain a sample; inembodiments, a sample may be contained in a sample collection device. Inembodiments, a device may be or comprise a cartridge configured tocontain a sample collection vessel.

In embodiments, a device may be or comprise a cartridge configured tocontain a reagent vessel, or a plurality of reagent vessels, and areaction vessel or a plurality of reaction vessels. In embodiments, sucha device may include reagents for use in nucleic acid assays; forimmunoassays (e.g., ELISA assays); general chemistry assays (e.g., forclinical electrolytes, vitamin levels, blood component levels, and othertargets); cytometric assays; and for combinations thereof. Inembodiments, such a device may include reagents and reaction vessels foruse in nucleic acid assays; for immunoassays (e.g., ELISA assays);general chemistry assays (e.g., for clinical electrolytes, vitaminlevels, blood component levels, and other targets); cytometric assays;and for combinations thereof. In embodiments, such a device may includereagents, reaction vessels, and tools, cuvettes, and other implementsfor use in nucleic acid assays; for immunoassays (e.g., ELISA assays);general chemistry assays (e.g., for clinical electrolytes, vitaminlevels, blood component levels, and other targets); cytometric assays;and for combinations thereof.

Accordingly, Applicants disclose systems for detecting the presence ofone or more of a plurality of markers indicative of an infectiousdisease in a small-volume clinical sample, comprising:

a) a sample handling system;

b) a detection station comprising an optical sensor;

c) a fluidically isolated sample collection unit configured to retain aclinical sample;

d) an assay station comprising at least a first and a second fluidicallyisolated assay unit, wherein the first unit comprises a first reagentand the second unit comprises a second reagent; and

e) a controller, wherein the controller comprises a local memory and isoperatively coupled to the sample handling system and the detectionstation;

wherein the system is configured to perform assays with one or both ofthe first and second assay units; wherein the local memory of thecontroller comprises a protocol comprising instructions for: i)directing the sample handling system to transfer a portion of theclinical sample to the first assay unit and to the second assay unit;and ii) directing the sample handling system to transfer the first assayunit and the second unit assay unit to the detection station.

Accordingly, Applicants disclose systems for detecting the presence ofone or more of a plurality of markers indicative of an infectiousdisease in a small-volume clinical sample, comprising:

a) a sample handling system;

b) a detection station comprising an optical sensor;

c) a fluidically isolated sample collection unit configured to retain aclinical sample;

d) an assay station comprising at least a first, second, and thirdfluidically isolated assay unit, wherein the first unit comprises afirst reagent, the second unit comprises a second reagent, and the thirdunit comprises a third reagent; and

e) a controller, wherein the controller comprises a local memory and isoperatively coupled to the sample handling system and the detectionstation;

wherein the system is configured to perform assays with any one or moreof the first, second, and third assay units; wherein the local memory ofthe controller comprises a protocol comprising instructions for: i)directing the sample handling system to transfer a portion of theclinical sample to the first assay unit, the second assay unit and thethird assay unit; and ii) directing the sample handling system totransfer the first assay unit, the second assay unit, and the thirdassay unit to the detection station. In embodiments, the system mayinclude only two assay units; or may include four assay units; or mayinclude more than four assay units.

In embodiments, the system is a point-of service system. In embodiments,the system is contained within a housing. In embodiments, the system islocated at a point-of-service location, and is configured for use inanalyzing a sample at said point-of-service location. In embodiments,the system is a point-of service system configured to perform aplurality of assays on a single small volume sample, or on aliquotsthereof.

Applicants further disclose systems for detecting the presence of one ormore of a plurality of markers indicative of an infectious disease in asmall-volume clinical sample, comprising:

-   -   a) a sample handling system;

b) a detection station comprising an optical sensor;

c) a fluid handling system configured to transport fluids betweencomponents of said system, wherein said transport of fluids comprisestransport of isolated aliquots of fluid;

d) a fluidically isolated sample collection unit configured to retain aclinical sample;

e) an assay station comprising at least a first, second, and thirdfluidically isolated assay unit, wherein the first unit comprises afirst reagent, the second unit comprises a second reagent, and the thirdunit comprises a third reagent; and

f) a controller, wherein the controller comprises a local memory and isoperatively coupled to the sample handling system and the detectionstation;

wherein the system is configured to perform assays with any one or moreof the first, second, and third assay units; wherein the local memory ofthe controller comprises a protocol comprising instructions for: i)directing the sample handling system to transfer a portion of theclinical sample to the first assay unit, the second assay unit and thethird assay unit; and ii) directing the sample handling system totransfer the first assay unit, the second assay unit, and the thirdassay unit to the detection station.

In embodiments, the system is a point-of service system. In embodiments,the system is contained within a housing. In embodiments, the fluidhandling system is configured to transport fluid within said housing. Inembodiments, the system is located at a point-of-service location, andis configured for use in analyzing a sample at said point-of-servicelocation. In embodiments, the system is a point-of service systemconfigured to perform a plurality of assays on a single small volumesample, or on aliquots thereof.

In embodiments, Applicants disclose a clinical sample processing system,comprising:

a) a sample handling system;

b) a detection station comprising an optical sensor;

c) a fluidically isolated sample collection unit configured to retain aclinical sample;

d) an assay station comprising at least a first, second, and thirdfluidically isolated assay unit, wherein the first unit comprises anantibody, the second unit comprises an oligonucleotide, and the thirdunit comprises a chromogen or a dye or other label; and

e) a controller, wherein the controller is operatively coupled to thesample handling system, wherein the sample handling system is configuredto transfer a portion of the clinical sample from the sample collectionunit to each of the first assay unit, the second assay unit, and thethird assay unit, and the device is configured to perform animmunoassay, a nucleic acid assay, and a general chemistry assaycomprising a chromogen. In embodiments, the system is a point-of servicesystem. In embodiments, the system is contained within a housing. Inembodiments, the system is located at a point-of-service location, andis configured for use in analyzing a sample at said point-of-servicelocation. In embodiments, the system is a point-of service systemconfigured to perform a plurality of assays on a single small volumesample, or on aliquots thereof.

In embodiments, Applicants disclose methods of performing at least 4different assays selected from immunoassays, nucleic acid assays,cytometric assays, and general chemistry assays on a small-volumeclinical sample, comprising:

a) introducing a clinical sample having a volume of no greater than 500microliters into a sample processing device, wherein the devicecomprises:

-   -   i) a sample handling system;    -   ii) a detection station;    -   iii) a cytometry station comprising an imaging device and a        stage for receiving a microscopy cuvette; and    -   iv) an assay station comprising at least a first, a second, a        third, and a fourth independently movable assay unit;

b) with the aid of the sample handling system, transferring a portion ofthe clinical sample to each of the first, second, third, and fourthassay units, wherein a different assay is performed in each of thefirst, second, third, and fourth assay units;

c) with the aid of the sample handling system, transferring the first,second, third, and fourth assay units to the detection station orcytometry station, wherein assay units comprising immunoassays orgeneral chemistry assays are transferred to the detection station andassay units comprising cytometric assays are transferred to thecytometry station;

d) with the aid of the detection station or cytometry station, obtainingdata measurements of the assay performed in each of the first, second,third, and fourth assay units.

In embodiments, the methods are point-of service methods. Inembodiments, the system used in performing the methods is containedwithin a housing. In embodiments, the methods are performed at apoint-of-service location, and may be used in analyzing a sample at saidpoint-of-service location. In embodiments, the methods comprise point-ofservice methods for performing a plurality of assays on a single smallvolume sample, or on aliquots thereof.

In embodiments, the methods include methods of determining the type ofinfection suffered by a subject. Methods of determining the type ofinfection as disclosed herein include, without limitation, methods asdisclosed herein comprising determining whether a subject suffers from abacterial, viral, yeast, fungus, and other infection. For example,methods of determining the type of infection as disclosed herein includemethods of determining whether a subject suffers from a bacterialinfection or from a viral infection. In embodiments, the methods includemethods of detecting, identifying, quantifying, and combinationsthereof, markers in a sample indicative of the type of infectionsuffered by a subject. Methods of detecting, identifying, quantifying,and combinations thereof, markers in a sample indicative of the type ofinfection as disclosed herein include, without limitation, methods asdisclosed herein comprising detecting, identifying, quantifying, andcombinations thereof, markers in a sample indicative of a bacterial,viral, yeast, fungus, and other infection. For example, methods asdisclosed herein include methods of detecting, identifying, quantifying,and combinations thereof, markers in a sample indicative of a bacterialinfection or a viral infection.

Methods disclosed herein may be used to determine whether a subjectsuffers from, e.g., a bacterial, viral, yeast, fungus, and otherinfection. Determination of the type of infection as disclosed hereinmay be used to guide therapy of the subject suffering from theinfection. Determination of the type of infection as disclosed hereinmay be used to guide selection of pharmaceuticals for treatment of thesubject suffering from the infection. Determination of the type ofinfection as disclosed herein may be used to guide selection of thedosage, or the dosing regimen, of pharmaceuticals used for the treatmentof the subject suffering from the infection. For example, methodsdisclosed herein may be used to determine whether a subject suffers froma bacterial infection or a viral infection. Determination of whether asubject suffers from a bacterial infection or a viral infection asdisclosed herein may be used to guide therapy of the subject sufferingfrom the infection. Determination of whether a subject suffers from abacterial infection or a viral infection as disclosed herein may be usedto guide selection of pharmaceuticals for treatment of the subjectsuffering from the infection. Determination of whether a subject suffersfrom a bacterial infection or a viral infection as disclosed herein maybe used to guide the selection of a pharmaceutical, selection of thedosage, the dosing regimen, or a combination thereof, used in thetreatment of the subject suffering from the infection. For example,where an infection is determined to be a bacterial infection,antibiotics may be prescribed; however, where an infection is determinedto be a viral infection, antibiotics are not indicated, and, inembodiments, will not be prescribed. Determination that a subjectsuffers from a viral infection may enable the subject to avoidunnecessary treatment and expense (e.g., where antibiotic therapy isavoided when the infection is identified as being a viral infection).Determination that a subject suffers from a viral infection may enablethe subject to obtain more appropriate therapy directed to viralinfections, as opposed to antibiotic therapy that is directed tobacterial infections.

Similarly, determination of whether a subject suffers from a yeast,fungus, and other infection, as opposed to a bacterial or viralinfection, may guide or determine the therapy provided to a subjectsuffering from an infectious disease, including guiding or determiningthe selection of a pharmaceutical, the selection of the dosage, thedosing regimen, or a combination thereof, used in the treatment of thesubject suffering from the infection. Determination of the type ofinfection may enable the subject to obtain more appropriate therapydirected to the particular type of infection suffered by the subject, asopposed to inappropriate, or less specific, therapy that may not be aseffective for that type of infection.

Accordingly, Applicants disclose herein methods for providing aprescription for treatment of an infectious disease in a subject,comprising: providing a clinical sample obtained from a subject;analyzing said clinical sample, wherein analyzing comprises testing for,or detecting the presence of, a plurality of disease markers, in theclinical sample; determining a suitable treatment for a diseaseindicated by the presence of a marker detected by said analysis; andproviding a prescription for said suitable treatment. Applicants furtherdisclose herein methods for providing a prescription for treatment of aninfectious disease in a subject, comprising: providing a clinical sampleobtained from a subject; analyzing said clinical sample at apoint-of-service (POS) location, wherein analyzing comprises testingfor, or detecting the presence of, a plurality of disease markers, inthe clinical sample; determining a suitable treatment for a diseaseindicated by the presence of a marker detected by said analysis; andproviding a prescription for said suitable treatment.

In embodiments of methods for providing a prescription for treatment ofan infectious disease in a subject, the analysis of the sample comprisesanalysis to determine whether the subject suffers from a bacterialinfection, a viral infection, a yeast infection, a fungal infection,other infection, or combination thereof. In embodiments of methods forproviding a prescription for treatment of an infectious disease in asubject, the analysis of the sample comprises analysis to determinewhether the subject suffers from a bacterial infection or a viralinfection. In embodiments of methods for providing a prescription fortreatment of an infectious disease in a subject, where the analysis ofthe sample determines that the subject suffers from a bacterialinfection, providing a prescription for said suitable treatmentcomprises prescription of an antibiotic. In embodiments of methods forproviding a prescription for treatment of an infectious disease in asubject, where the analysis of the sample determines that the subjectsuffers from a viral infection, providing a prescription for saidsuitable treatment comprises avoiding the prescription of an antibiotic,and may include the prescription of an anti-viral drug. In embodimentsof methods for providing a prescription for treatment of an infectiousdisease in a subject, where the analysis of the sample determines thatthe subject suffers from a fungal infection, providing a prescriptionfor said suitable treatment comprises the prescription of an anti-fungaldrug. In embodiments of methods for providing a prescription fortreatment of an infectious disease in a subject, where the analysis ofthe sample determines that the subject suffers from a bacterialinfection that is a mycoplasma infection, providing a prescription forsaid suitable treatment comprises the prescription of ananti-mycoplasmal drug. In embodiments of methods for providing aprescription for treatment of an infectious disease in a subject, wherethe analysis of the sample determines that the subject suffers from ayeast infection, providing a prescription for said suitable treatmentcomprises the prescription of an anti-yeast drug.

Accordingly, the systems, devices, and methods disclosed herein arepoint-of service methods. In embodiments, the systems disclosed herein,including the systems used in performing the methods disclosed herein,may be contained and the methods performed within a housing. Inembodiments, the devices disclosed herein may be placed or used within ahousing, e.g., a housing containing a system disclosed herein. Inembodiments, the systems disclosed herein may be located at a POSlocation, and the methods may be performed at a point-of-servicelocation. The systems and methods disclosed herein may be used inanalyzing a sample at said point-of-service location. In embodiments,the systems and methods comprise point-of service methods for performinga plurality of assays on a single small volume sample, or on aliquotsthereof.

In embodiments, Applicants disclose systems, methods, and devices fordetecting one or more of a plurality of markers indicative of a diseasein a clinical sample obtained at a point-of-service (POS) location. Inembodiments, such a clinical sample is a small volume clinical sample.In embodiments, the one or more markers are detected in a short periodof time. In embodiments, the sample is obtained at a POS location. Inembodiments, the systems and devices are located at a POS location. Inembodiments, the detection of the one or more markers is performed at aPOS location. In embodiments, the diseases are infectious diseases. Inembodiments, the diseases are caused by a disease-causing agent selectedfrom the group of disease-causing organisms consisting of a virus, abacterium (including a mycoplasma), a fungus, a yeast, and othermicro-organisms. In embodiments, the diseases are infectious respiratorydiseases, and may be upper respiratory diseases, and may be lowerrespiratory diseases.

Accordingly, in embodiments, Applicants disclose POS systems, methods,and devices. In embodiments, such POS systems, methods, and devicescomprise automated POS systems, methods, and devices. In embodiments,for example, Applicants disclose an automated POS system, automatedmethods, and devices thereof, for detecting one or more of a pluralityof markers indicative of a disorder in a clinical sample obtained at aPOS location; such a disorder may be a respiratory disorder, and may bea disorder caused by a disease-causing agent selected from the group ofdisease-causing organisms consisting of a virus, a bacterium (includinga mycoplasma), a fungus, a yeast, and other micro-organisms. Inembodiments, such automated POS systems, automated methods, and devicesthereof, are configured for use at POS locations, and for use withsamples obtained at POS locations. In embodiments, such automated POSsystems, automated methods, and devices thereof, are configured for useon a single small-volume clinical sample. In embodiments, such automatedPOS systems, automated methods, and devices thereof, are configured todetect, if present, one or more of a plurality of markers indicative ofa disorder in a clinical sample in a short period of time.

In embodiments, such automated POS systems, methods, and devices arelocated at a POS location selected from a retail pharmacy, asupermarket, a clinic, a hospital, and a doctor's office. Inembodiments, a prescription for a treatment is issued at said POSlocation. In embodiments, a prescription for a treatment is issued atsaid POS location pursuant to the results of such testing performed byan automated POS systems, methods, and devices located at the POSlocation. In embodiments, a prescription for a treatment is filled atsaid POS location, wherein the prescription was issued for a treatmentis pursuant to the results of such testing performed by an automated POSsystems, methods, and devices located at the POS location. Inembodiments, a bill for testing is issued at the POS location; such abill may be issued automatically. In embodiments, a bill for aprescription is issued at the POS location, wherein the prescription wasissued for a treatment is pursuant to the results of such testingperformed by an automated POS systems, methods, and devices located atthe POS location; such a bill may be issued automatically. Inembodiments, a bill for a test or a prescription may be issued from thePOS location to a subject's insurance carrier; such a bill may be issuedautomatically. In embodiments, an automatic payment may be made for atest performed or a prescription filled at from the POS location to asubject's insurance carrier, pursuant to a bill issued automaticallyfrom the POS location.

Accordingly, Applicants disclose devices configured to measure or detecta disease-causing agent or marker indicative of a disease-causing agentin a sample according to a method disclosed herein. Such a sample may bea small-volume clinical sample. Such devices may be configured tomeasure or detect a particular disease-causing agent or markerindicative of a particular disease-causing agent in a sample in lessthan about three hours, or less than about two hours, or in less thanabout one hour, or, in embodiments, in less than about 40 minutes, or inless than about 30 minutes.

Devices disclosed herein may be configured to perform an assay for thedetection or measurement of a plurality of disease-causing agents ormarkers indicative thereof. Devices disclosed herein may be configuredto perform an assay for the detection or measurement of a particulardisease-causing agent or marker indicative thereof and also to performan assay comprising the measurement of a morphological characteristic ofa cell in the sample. Devices disclosed herein may be configured toperform an assay for the measurement of a disease-causing agent ormarker indicative thereof and also to perform an assay comprising themeasurement of another analyte, e.g., a cytokine, a prostaglandin,histamine, a steroid (e.g., a glucocorticoid, or other steroid), avitamin, a hormone, a drug or metabolite of a drug, or other analyte.Such devices may be configured wherein the assays, or the order ofperformance of assays, that are performed by said device may be alteredby communication with another device.

Methods and compositions disclosed herein provide rapid assays whichrequire only small amounts of sample, such as only small amounts ofsaliva, urine, blood, or fluid in which a throat swab, cheek swab, ornasal swab has been immersed. In embodiments, a plurality of samples,including a plurality of small samples, may comprise a plurality ofsample types, such as saliva, urine, blood, or fluid samples, may beprovided to, and analyzed by, systems, devices, and methods disclosedherein. Methods, devices and systems disclosed herein are configured toperform such rapid assays which require only small amounts of sample.Methods, devices and systems disclosed herein are configured to performsuch rapid assays on a plurality of sample types, and may require onlysmall amounts of each sample type. Methods, devices and systemsdisclosed herein are configured to perform multiple assays on a sample,or on a plurality of sample types, and may be used to screen for one ormore of a plurality of diseases. Methods, devices and systems disclosedherein are configured to perform multiple assays on a sample, or on aplurality of sample types, and may be used to screen for diseases causedby one or more of viruses, bacteria, yeast, fungus, mycoplasma, archea,fungus, yeast, parasites, and other micro-organisms. Accordingly, themethods, devices, and systems disclosed herein provide rapid tests,which require only small clinical samples, and thus provide advantagesover other methods, devices, and systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a graphic summary of the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for a range of markers and for twodifferent concentration ranges of the markers (10 c/μl and 100 c/μl,where “c/μl” means copies per microliter (μL)). The times are labeled“LOD” (“length of delay”). The vertical axis is shown in units ofrelative fluorescence (relative fluorescence units, RFU), in thousands.

FIG. 1B provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers for variousdiseases (at 100 c/μl).

FIG. 1C provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers for severalinfluenza strains and identifying targets (at 100 c/μl).

FIG. 1D provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers of severalrespiratory diseases (at 100 c/μl).

FIG. 1E provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers for severalsexually transmitted diseases (at 100 c/μl).

FIG. 1F provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers for severaldiseases that can be detected in blood (at 100 c/μl).

FIG. 2A shows amplification over time, showing detection of influenza A(seasonal H1N1 strain) marker at times well before any significantamounts of amplification of non-target nucleic acid message occurred.The horizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 2B shows the limit of detection of influenza A (seasonal H1N1strain) in a sample. The height of the bars indicates the time until thecopy number shows an inflection (rises significantly above thebackground level), with the horizontal axis indicating the initialnumbers of copies of influenza A (seasonal H1N1 strain) message; “NTC”indicates “no template control” (no added copies of the target marker).FIG. 2B shows detection of influenza A (seasonal H1N1 strain) marker attimes well before any significant amounts of amplification of non-targetnucleic acid message occurred.

FIG. 3A shows amplification over time, showing detection of influenza A(novel H1N1 strain) marker at times well before any significant amountsof amplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 3B shows the limit of detection of influenza A (novel H1N1 strain)in a sample. The height of the bars indicates the time until the copynumber shows an inflection (rises significantly above the backgroundlevel), with the horizontal axis indicating the initial numbers ofcopies of influenza A (novel H1N1 strain) message; “NTC” indicates “notemplate control” (no added copies of the target marker). FIG. 3B showsdetection of influenza A (novel H1N1 strain) marker at times well beforeany significant amounts of amplification of non-target nucleic acidmessage occurred.

FIG. 4A shows amplification over time, showing detection of influenza A(H3N2 strain) marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 4B shows the limit of detection of influenza A (H3N2 strain) in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating the initial numbers of copies ofinfluenza A (H3N2 strain) message; “NTC” indicates “no template control”(no added copies of the target marker). FIG. 4B shows detection ofinfluenza A (H3N2 strain) marker at times well before any significantamounts of amplification of non-target nucleic acid message occurred.

FIG. 5A shows amplification over time, showing detection of influenza A(H7N9 strain) marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 5B shows the limit of detection of influenza A (H7N9 strain) in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating the initial numbers of copies ofinfluenza A (H7N9 strain) message; “NTC” indicates “no template control”(no added copies of the target marker). FIG. 5B shows detection ofinfluenza A (H7N9 strain) marker at times well before any significantamounts of amplification of non-target nucleic acid message occurred.

FIG. 6A shows amplification over time, showing detection of influenza A(H5N1 strain) marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 6B shows the limit of detection of influenza A (H5N1 strain) in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating the initial numbers of copies ofinfluenza A (H5N1 strain) message; “NTC” indicates “no template control”(no added copies of the target marker). FIG. 6B shows detection ofinfluenza A (H5N1 strain) marker at times well before any significantamounts of amplification of non-target nucleic acid message occurred.

FIG. 7A shows amplification over time, showing detection of influenza Bmarker at times well before any significant amounts of amplification ofnon-target nucleic acid message occurred. The horizontal axis isdenominated in “cycles” although no cycling of temperature was used;each unit of “cycles” is approximately one minute, so that the numbersof the horizontal axis may be read in terms of minutes. The verticalaxis is shown in units of relative fluorescence (relative fluorescenceunits, RFU), in thousands.

FIG. 7B shows the limit of detection of influenza B in a sample. Theheight of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thehorizontal axis indicating the initial numbers of copies of influenza Bmessage; “NTC” indicates “no template control” (no added copies of thetarget marker). FIG. 7B shows detection of influenza B marker at timeswell before any significant amounts of amplification of non-targetnucleic acid message occurred.

FIG. 8A shows amplification over time, showing detection of influenzaMatrix Protein marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 8B shows the limit of detection of influenza Matrix Protein markerin a sample. The height of the bars indicates the time until the copynumber shows an inflection (rises significantly above the backgroundlevel), with the horizontal axis indicating the initial numbers ofcopies of influenza Matrix Protein message; “NTC” indicates “no templatecontrol” (no added copies of the target marker). FIG. 8B shows detectionof influenza Matrix Protein marker at times well before any significantamounts of amplification of non-target nucleic acid message occurred.

FIG. 9A shows amplification over time, showing detection of atuberculosis marker (Myobacterium tuberculosis) at times well before anysignificant amounts of amplification of non-target nucleic acid messageoccurred. The horizontal axis is denominated in “cycles” although nocycling of temperature was used; each unit of “cycles” is approximatelyone minute, so that the numbers of the horizontal axis may be read interms of minutes. The vertical axis is shown in units of relativefluorescence (relative fluorescence units, RFU), in thousands.

FIG. 9B shows the limit of detection of tuberculosis in a sample. Theheight of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thenumbers “TB 1000” indicating 1000 copies of tuberculosis marker message,“TB 100” indicating 100 copies of tuberculosis marker message, and “TB10” indicating 10 copies of tuberculosis marker message; “NTCs”indicates “no template controls” (no added copies of the target marker).

FIG. 10A shows amplification over time, showing detection of astaphylocccus marker (Staphylococcus aureus) at times well before anysignificant amounts of amplification of non-target nucleic acid messageoccurred. The horizontal axis is denominated in “cycles” although nocycling of temperature was used; each unit of “cycles” is approximatelyone minute, so that the numbers of the horizontal axis may be read interms of minutes. The vertical axis is shown in units of relativefluorescence (relative fluorescence units, RFU), in thousands.

FIG. 10B shows the limit of detection of Staphylococcus aureus in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating numbers of copies of Staphylococcusaureus message; “NTC” indicates “no template control” (no added copiesof the target marker).

FIG. 11A shows amplification over time, showing detection of astaphylocccus marker (Methicillin-Resistant Staphylococcus aureus—MRSA)at times well before any significant amounts of amplification ofnon-target nucleic acid message occurred. The horizontal axis isdenominated in “cycles” although no cycling of temperature was used;each unit of “cycles” is approximately one minute, so that the numbersof the horizontal axis may be read in terms of minutes. The verticalaxis is shown in units of relative fluorescence (relative fluorescenceunits, RFU), in thousands.

FIG. 11B shows the limit of detection of MRSA Staphylococcus aureus in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating numbers of copies of MRSAStaphylococcus aureus message; “NTC” indicates “no template control” (noadded copies of the target marker).

FIG. 12A shows amplification over time, showing detection of astreptococcus marker (Streptococcus Group A) at times well before anysignificant amounts of amplification of non-target nucleic acid messageoccurred. The horizontal axis is denominated in “cycles” although nocycling of temperature was used; each unit of “cycles” is approximatelyone minute, so that the numbers of the horizontal axis may be read interms of minutes. The vertical axis is shown in units of relativefluorescence (relative fluorescence units, RFU), in thousands.

FIG. 12B shows the limit of detection of Streptococcus Group A in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating numbers of copies of StreptococcusGroup A message; “NTC” indicates “no template control” (no added copiesof the target marker).

FIG. 13A shows amplification over time, showing detection of aBordetella pertussis marker at times well before any significant amountsof amplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 13B shows detection of Bordetella pertussis in a sample. The heightof the bars indicates the time until the copy number shows an inflection(rises significantly above the background level), with the horizontalaxis indicating numbers of copies of Bordetella pertussis message (asfinal DNA copy per μL); “NTC” indicates “no template control” (no addedcopies of the target marker).

FIG. 14A shows amplification over time, showing detection of anadenovirus B marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 14B shows the limit of detection of Adenovirus B in a sample. Theheight of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thehorizontal axis indicating numbers of copies of Adenovirus B message;“NTC” indicates “no template control” (no added copies of the targetmarker).

FIG. 15A shows amplification over time, showing detection of anadenovirus C marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 15B shows the limit of detection of Adenovirus C in a sample. Theheight of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thehorizontal axis indicating numbers of copies of Adenovirus C message;“NTC” indicates “no template control” (no added copies of the targetmarker).

FIG. 16A shows amplification over time, showing detection of anadenovirus E marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 16B shows the limit of detection of Adenovirus E in a sample. Theheight of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thehorizontal axis indicating numbers of copies of Adenovirus E message;“NTC” indicates “no template control” (no added copies of the targetmarker).

FIG. 17A shows amplification over time, showing detection of a HerpesSimplex Virus (HSV) marker at times well before any significant amountsof amplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 17B shows the limit of detection of Herpes Simplex Virus (HSV) in asample. The height of the bars indicates the time until the copy numbershows an inflection (rises significantly above the background level),with the horizontal axis indicating numbers of copies of HSV message;“NTC” indicates “no template control” (no added copies of the targetmarker).

FIG. 18A shows amplification over time, showing detection of a Treponemapallidum marker at times well before any significant amounts ofamplification of non-target nucleic acid message occurred. Thehorizontal axis is denominated in “cycles” although no cycling oftemperature was used; each unit of “cycles” is approximately one minute,so that the numbers of the horizontal axis may be read in terms ofminutes. The vertical axis is shown in units of relative fluorescence(relative fluorescence units, RFU), in thousands.

FIG. 18B shows the limit of detection of Treponema pallidum in a sample.The height of the bars indicates the time until the copy number shows aninflection (rises significantly above the background level), with thehorizontal axis indicating numbers of copies of Treponema pallidummessage; “NTC” indicates “no template control” (no added copies of thetarget marker).

FIG. 19A shows amplification over time, the rise in relativefluorescence at about 15 to 20 minutes indicating the presence of anInfluenza H1N1 seasonal marker. The horizontal axis is in “minutes; thevertical axis is shown in units of relative fluorescence (relativefluorescence units, RFU).

FIG. 19B shows amplification of “no template control” (no added copiesof the target marker; NTC). Note that most experiments showed noamplification; the three runs that show late increases in relativefluorescence did so at about 25 minutes or later. The horizontal axis isin “minutes; the vertical axis is shown in units of relativefluorescence (relative fluorescence units, RFU).

FIG. 20A shows an exemplary vessel for holding a swab (a swab vessel)and an exemplary cartridge (which includes cavities and wells forreagents and vessels, and is configured to hold reagent vessels,reaction vessels, and other vessels and implements). Arrows leading awayfrom the swab vessel indicate how the swab vessel may be placed into areceptacle in the cartridge.

FIG. 20B shows an exemplary swab vessel (configured for holding a swab)and an exemplary cartridge (which includes cavities and wells forreagents and vessels, and is configured to hold reagent vessels,reaction vessels, and other vessels and implements). In addition to thecavities and wells configured to hold reagent vessels, reaction vessels,and other vessels and implements as shown in the embodiment of FIG. 20A,the exemplary cartridge shown in FIG. 20B includes cavities and wellssuitable for holding other sample vessels, e.g., blood or urine samplevessels, in addition to swab vessels. Arrows leading away from the swabvessel indicate how the swab vessel may be placed into a receptacle inthe cartridge.

FIG. 20C shows an exemplary swab vessel, and an exemplary cartridgewhich includes cavities and wells for holding a swab and a swab vessel,as well as cavities and wells configured to hold reagent vessels,reaction vessels, and other vessels and implements (which may optionallyinclude other sample vessels, e.g., blood or urine sample vessels).Arrows leading away from the swab indicate how the swab may be placedinto a swab receptacle in the cartridge. Arrows leading away from theswab vessel indicate how the swab vessel may be placed into a swabvessel receptacle in the cartridge.

FIG. 21 shows examples of swabs which may be used to obtain samples fromthe throat, nasal passages, cheeks, or other body locations of subjects.

FIG. 22 shows various panels naming disorders which may be identified bythe methods and devices discussed herein.

FIG. 23A shows various influenza panels naming influenza types which maybe identified by the methods and devices discussed herein.

FIG. 23B shows inflection times for several influenza types which may beidentified by the methods and devices discussed herein.

FIG. 24A shows various respiratory disease panels naming respiratorydisease types which may be identified by the methods and devicesdiscussed herein.

FIG. 24B shows inflection times for upper and lower respiratory tractdisease types which may be identified by the methods and devicesdiscussed herein.

FIG. 25A shows various hospital acquired infectious disease panelsnaming respiratory disease types which may be identified by the methodsand devices discussed herein.

FIG. 25B shows inflection times for various hospital acquired infectiousdisease panels naming respiratory disease types which may be identifiedby the methods and devices discussed herein.

FIG. 26 shows results of an assay for influenza A that is designed to beinclusive for all Influenza A subtypes. The results are specific.

FIG. 27 shows the specificity of the nucleic acid assays for the targetH2N2 influenza type.

FIG. 28 shows the specificity of the nucleic acid assays for the targetH1N1 seasonal influenza type.

FIG. 29 lists potential interfering substances for the sexuallytransmitted disease (STD) panel that were found not to interfere withthe nucleic acid assays.

FIG. 30 lists potential interfering substances for the sexuallytransmitted disease (STD) urine panel that were found not to interferewith the nucleic acid assays.

FIG. 31 lists potential interfering substances for the blood panel thatwere found not to interfere with the nucleic acid assays.

DETAILED DESCRIPTION

Description and disclosure of examples of reagents, assays, methods,kits, devices, and systems which may be used with the methods, assays,reagents, devices and systems disclosed herein may be found, forexample, in U.S. Pat. No. 8,088,593; U.S. Pat. No. 8,380,541; U.S.patent application Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/769,820, filed Feb. 18, 2013; PCT/US2012/57155,filed Sep. 25, 2012; U.S. patent application Ser. No. 13/244,949, filedSep. 26, 2011; U.S. Application Ser. No. 61/766,095, filed Feb. 18,2013; U.S. Application Ser. No. 61/673,245, filed Sep. 26, 2011; U.S.Patent Application Ser. No. 61/874,976, filed Sep. 6, 2013; U.S. PatentApplication Ser. No. 61/885,462, filed Oct. 1, 2013; U.S. PatentApplication Ser. No. 62/001,039, filed May 20, 2014; U.S. PatentApplication Ser. No. 62/001,053, filed May 21, 2014; U.S. PatentApplication Ser. No. 62/010,382, filed Jun. 10, 2014; U.S. PatentApplication Ser. No. 61/885,467, filed Oct. 1, 2013; U.S. PatentApplication Ser. No. 61/879,664, filed Sep. 18, 2013; and U.S. PatentApplication 61/805,923, filed Mar. 27, 2013, the disclosures of whichpatents and patent applications are all hereby incorporated by referencein their entireties.

Disclosure of methods of detecting nucleic acid targets include, forexample, methods, assays, reagents, and devices as disclosed in U.S.Application Ser. No. 61/800,606, filed Mar. 15, 2013; U.S. ApplicationSer. No. 61/908,027, filed Nov. 22, 2013; U.S. Application Ser. No.62/001,050, filed May 20, 2014; U.S. application Ser. No. 14/214,850,filed Mar. 15, 2014; PCT/US2014/030034, filed Mar. 15, 2014; U.S.Application Ser. No. 61/800,241, filed Mar. 15, 2013; and U.S.Application Ser. No. 61/800,340, filed Mar. 15, 2013; the disclosures ofwhich patent applications are hereby incorporated by reference in theirentireties. Further methods for the detection of nucleic acid targetsinclude, for example, Polymerase Chain Reaction (PCR) methods described,for example, in U.S. Pat. No. 4,683,195; and generally in Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); Erlich, ed., PCRTechnology (Stockton Press, N Y, 1989).

Disclosure of methods for detecting protein targets, including antibodymethods, may be found, for example, in U.S. Pat. No. 4,376,110; U.S.Pat. No. 4,816,567; U.S. Pat. No. 7,429,652; European Patent EP 404,097;and International Patent Application Publication WO 93/11161, thedisclosures of which are hereby incorporated by reference in theirentireties. Further methods for the detection of protein targets(generically termed “immunoassays” herein) include, for example, director competitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays.

Disclosure regarding systems, devices, and methods for analyzingclinical samples, including clinical samples such as small-volumeclinical samples, and including systems, devices, and methods foranalyzing small-volume clinical samples in short periods of time, may befound, for example, in U.S. Pat. No. 8,380,541; U.S. Pat. No. 8,088,593;U.S. Pat. No. 8,380,541; U.S. patent application Ser. No. 13/769,798,filed Feb. 18, 2013; U.S. patent application Ser. No. 13/769,820, filedFeb. 18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb.18, 2013; PCT/US2012/57155, filed Sep. 25, 2012; U.S. Patent Application61/805,923, filed Mar. 27, 2013; and incorporated by reference herein(supra).

Before the present novel target-binding molecules, compositions, assays,methods, and kits are disclosed and described, it is to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting. It isalso to be understood that the present disclosure provides explanatoryand exemplary descriptions and examples, so that, unless otherwiseindicated, the molecules, compositions, assays, methods, and kitsdisclosed herein are not limited to the specific embodiments describedherein.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a salt” refers to a single salt or mixtures of differentsalts, and the like.

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Acronyms and abbreviations, such as “rpm” (revolutions per minute),“min” (minute), “sec” (second), and so forth, have their customarymeanings.

As used herein, the terms “normal” and “normal level” refer to thelevels of a marker as found in a healthy population of normal subjects.For example, the normal level for a particular type of white blood cellfound is the level of that type of white blood cell found in bloodsamples from a healthy population of normal subjects.

As used herein, the terms “high” and “high level” and the like refer tolevels that significantly exceed normal levels, that is, a high level ofa marker is one that significantly exceeds the levels of that markerthat is found in a healthy population of normal subjects.

As used herein, the terms “low” and “low level” and the like refer tolevels that are below normal levels, that is, a low level of a marker isone that is below the levels of that marker that is found in a healthypopulation of normal subjects.

It will be understood that, where a marker is typically absent, orscarce, in normal subjects, a normal level of a marker may be very lowin absolute numbers (e.g., as measured by numbers of markers per unitvolume, or weight of marker per unit volume), and still be the normallevel for that marker. Thus, for example, where the marker is anantibody to a particular infectious disease, and most healthy normalsubjects have not been recently exposed to that particular disease, thenormal levels of antibodies to that disease may be low in absoluteterms, and levels in a subject that exceed the normal level wouldindicate that the subject has recently been exposed to, or is sufferingan infection by, that disease.

The term “isolated” as used herein when used to describe the variousnucleic acids and proteins disclosed herein, means the nucleic acid orprotein (or other molecule) has been separated and/or recovered from atleast one contaminant with which it is ordinarily associated.Ordinarily, however, isolated nucleic acids and proteins will beprepared by at least one purification step.

The term “moiety” as used herein refers to any particular composition ofmatter, e.g., a molecular fragment, an intact molecule, or a mixture ofmaterials.

As used herein, the terms “disease-causing agent”, “disease-causingorganism” and plurals and grammatical equivalents are usedinterchangeably to refer to viruses, bacteria, yeast, and othermicro-organisms which may cause disease in a subject. Thus, whenreferring to diseases and their causes, the terms “agent”, “organism”and plurals and grammatical equivalents are used interchangeably herein.

As used herein, the term “virus” refers to organisms which includenucleic acid message (either RNA or DNA) which allows their replicationin infected host cells. The term virus includes both DNA viruses or RNAviruses. Viruses may cause diseases.

As used herein, the term “micro-organism” refers to small unicellular ormulticellular organisms which may infect cells, organs, tissues, orsurfaces of plants or animals, including humans. The term microorganismincludes bacteria (including mycoplasma), archea, fungus, yeast,parasites, and other small organisms. Micro-organisms may causediseases.

As used herein, the term “bacteria” refers to small unicellular,prokaryotic organisms which may infect cells, organs, tissues, orsurfaces of plants or animals, including humans. The term bacteriaincludes Gram negative bacteria and Gram positive bacteria. Bacteria maycause diseases. Mycoplasma are a form of bacteria that lack cell walls.

As used herein, the term “drug” is used broadly to refer to any agentwhich may be administered to a subject for the purpose of treating adisease or condition suffered by the subject; such treating may includeprevention, amelioration of symptoms, hastening recovery, strengtheningthe patient in the face of a disease or condition, as well as directlycombating the disease or condition. Where the disease or conditionresults from an infection, e.g., is due to an infectious disease, thedrug may be, without limitation, an antibiotic, an anti-viral drug, ananti-fungal drug, an anti-mycoplasmal drug, an anti-yeast drug, orcombinations thereof.

As used herein, the term “antibiotic” is used broadly to refer to drugswhich act to reduce or eliminate bacterial infections. Antibioticsinclude, without limitation, penicillin, ampicillin, amoxicillin,tetracycline, oxytetracycline, doxycycline, minocycline, a sulfonamidesulfa-drug (such as, e.g., sulfanilamide, sulfamethoxazole,sulfadiazine), erythromycin, ciprofloxacin, gentamycin, oligomycin,azithromycin, clarithromycin, a cephalosporin, e.g., cefaclor,cefprozil, cefuroxime axetil, loracarbef, cefdinir, cefixime,cefpodoxime proxetil, ceftibuten, or ceftriaxone, gramicidin,valinomycin, nonactin, alamethicin, and other antibiotics.

As used herein, the term “anti-mycoplasma” is used broadly to refer todrugs which act to reduce or eliminate bacterial infections where thebacteria are mycoplasma. Antibiotics that target the cell wall aretypically ineffective against mycoplasma, which lack cell walls.Antibiotics such as, e.g., plasmocin, doxocycline, minocycline,gramicidin, valinomycin, nonactin, alamethicin, macrolide antibiotics,and others may be used to treat mycoplasmal infections.

As used herein, the term “anti-viral” is used broadly to refer to drugswhich act to reduce or eliminate viral infections. Ant-viral drugsinclude, for example, zanamivir, oseltamivir, acyclovir, adefovir,darunivir, famciclovir, ganciclovir, nexavir, rifampicin, pieconaril,amantadine, rimantadine, and others.

As used herein, the term “anti-fungal” is used broadly to refer to drugswhich act to reduce or eliminate fungus infections. Ant-fungal drugsinclude, for example, amphotericin, nystatin, candicin, filipin,hamycin, netamycin, rimocydin, bifonazole, clotrimazole, otherimidazole, triazole, and thiazoles, and others. Some drugs which may beused to treat fungal infections may also be suitable for treating yeastinfections.

As used herein, the term “anti-yeast” is used broadly to refer to drugswhich act to reduce or eliminate yeast infections. Anti-yeast drugsinclude, for example, antimycotics such as, e.g., clotrimazole,nystatin, fluconazole, ketoconazole, amphotericin, gentian violet, andother drugs. Some drugs which may be used to treat yeast infections mayalso be suitable for treating fungal infections.

As used herein, the phrase “nucleic acid markers indicative of” aparticular infection refers to nucleic acid molecules (includingsingle-stranded and double-stranded DNA and RNA molecules) and fragmentsthereof, which are derived from disease-causing organisms, or are copiesof, or are substantially similar to, or are complementary to, nucleicacid molecules derived from the organism which causes that particularinfectious disease. Detection of nucleic acid markers indicative of aparticular infection in a sample indicates that the disease-causingorganism is, or was, present in the sample and thus that the subject hasbeen exposed to the disease-causing organism, and likely suffers orsuffered from the particular infection caused by that particulardisease-causing organism.

As used herein, the phrase “antibody markers indicative of” a particularinfection refers to antibodies (or portions or fragments thereof) whichare directed to an antigen or antigens found on the organisms that causethat particular infectious disease. Detection of antibody markersindicative of a particular infection in a sample indicates that thedisease-causing organism is, or was, present in the sample and thus thatthe subject has been exposed to the disease-causing organism, and likelysuffers or suffered from the particular infection caused by thatparticular disease-causing organism.

As used herein, a nucleic acid comprises a molecule made up ofnucleotides, and refers to deoxyribonucleic acid (DNA) and toribonucleic acid (RNA) molecules.

As used herein, “nucleic acid” includes both DNA and RNA, including DNAand RNA containing non-standard nucleotides. A “nucleic acid” containsat least one polynucleotide (a “nucleic acid strand”). A “nucleic acid”may be single-stranded or double-stranded. The term “nucleic acid”refers to nucleotides and nucleosides which make up, for example,deoxyribonucleic acid (DNA) macromolecules and ribonucleic acid (RNA)macromolecules. Nucleic acids may be identified by the base attached tothe sugar (e.g., deoxyribose or ribose); as used herein, the followingabbreviations for these bases are used to represent nucleic acids insequence listings identifying and describing their structures (eitherupper-case or lower-case may be used).

TABLE 1A Base (in Nucleic Acid) Letter Code Adenine A Thymine T GuanineG Cytosine C Uracil U

As used herein, a “polynucleotide” refers to a polymeric chaincontaining two or more nucleotides. “Polynucleotides” includes primers,oligonucleotides, nucleic acid strands, etc. A polynucleotide maycontain standard or non-standard nucleotides. Typically, apolynucleotide contains a 5′ phosphate at one terminus (“5′ terminus”)and a 3′ hydroxyl group at the other terminus (“3′ terminus) of thechain. The most 5′ nucleotide of a polynucleotide may be referred toherein as the “5′ terminal nucleotide” of the polynucleotide. The most3′ nucleotide of a polynucleotide may be referred to herein as the “3′terminal nucleotide” of the polynucleotide.

As used herein, a “target” nucleic acid or molecule refers to a nucleicacid of interest. A target nucleic acid/molecule may be of any type,including single-stranded or double stranded DNA or RNA (e.g. mRNA).

As used herein, a nucleic acid molecule which is described as containingthe “sequence” of a template or other nucleic acid may also beconsidered to contain the template or other nucleic acid itself (e.g. amolecule which is described as containing the sequence of a template mayalso be described as containing the template), unless the contextclearly dictates otherwise.

As used herein, “complementary” sequences refer to two nucleotidesequences which, when aligned anti-parallel to each other, containmultiple individual nucleotide bases which pair with each other. It isnot necessary for every nucleotide base in two sequences to pair witheach other for sequences to be considered “complementary”. Sequences maybe considered complementary, for example, if at least 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of thenucleotide bases in two sequences pair with each other. In addition,sequences may still be considered “complementary” when the total lengthsof the two sequences are significantly different from each other. Forexample, a primer of 15 nucleotides may be considered “complementary” toa longer polynucleotide containing hundreds of nucleotides if multipleindividual nucleotide bases of the primer pair with nucleotide bases inthe longer polynucleotide when the primer is aligned anti-parallel to aparticular region of the longer polynucleotide.

As used herein, a “concatemer” refers to a nucleic acid molecule whichcontains within it two or more copies of a particular nucleic acid,wherein the copies are linked in series. Within the concatemer, thecopies of the particular nucleic acid may be linked directly to eachother, or they may be indirectly linked (e.g. there may be nucleotidesbetween the copies of the particular nucleic acid). In an example, theparticular nucleic acid may be that of a double-stranded nucleic acidtemplate, such that a concatemer may contain two or more copies of thedouble-stranded nucleic acid template. In another example, theparticular nucleic acid may be that of a polynucleotide template, suchthat a concatemer may contain two or more copies of the polynucleotidetemplate.

As used herein, a “saccharide” is a molecule comprising one, a few, ormultiple sugar moieties, and includes monosaccharides, oligosaccharides,and polysaccharides.

As used herein, a protein comprises a molecule made up of amino acids,the amino acids covalently linked by amide bonds. The terms “peptide”,“polypeptide” and “protein” may be used interchangeably to refer tomolecules comprised of amino acids linked by peptide bonds. Individualamino acids may be termed “residues” of a polypeptide or protein. Theamino acid sequences of polypeptides disclosed herein may be identifiedby SEQ ID NO: presented as a string of letters, where the letters havethe following meanings:

TABLE 1B 3-Letter 1-Letter AminoAcid Code Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acidGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

As used herein, a “cytokine” is a naturally occurring protein moleculesoften released in mammals in response to injury, infection,inflammation, or other stressor. Cytokines include lymphokines,interleukins, chemokines, interferons, and other cytokines. Cytokinesmay be inflammatory cytokines (tending to cause inflammation; alsotermed pro-inflammatory cytokines) or may be anti-inflammatory cytokines(tending to suppress inflammation). Inflammatory cytokines include, forexample, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12(IL-12), interleukin-18 (IL-18), gamma interferon (IFN-γ), and tumornecrosis factor alpha (TNF-α). Anti-inflammatory cytokines include, forexample, interleukin-10 (IL-10). Some cytokines may have multipleactions or effects (e.g., interleukin-6 (IL-6) has both inflammatory andanti-inflammatory effects).

As used herein, the terms “marker of inflammation”, “inflammatorymarker”, and plurals and grammatical equivalents thereof refer tomarkers which may be detected in a sample, and which may be identifiedin a sample, which indicate the presence of, or level of, inflammationin the subject from which the sample was obtained. Markers ofinflammation include both peptide and non-peptide markers; for example,markers of inflammation include, without limitation, prostaglandins,tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interferon gamma (IF-γ), bradykinin,complement system molecules, blood-clotting factors, C-reactive protein,erythrocyte sedimentation rate (ESR), white blood cell count, andmorphological changes in blood and other cells.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, andantibody fragments, and includes human an humanized antibodies.Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. The most abundant classof antibodies is the IgG class, characterized by having molecularweights of about 150 kD.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. For example, monoclonal antibodies may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567 to Cabilly et al.).

The term “intact antibody” refers to the complete antibody, or the aminoacid sequence of the complete antibody, of which an antibody fragment isa part. It will be understood that an antibody fragment may be producedby partial digestion (e.g., by papain or pepsin) of an intact antibody,or may be produced by recombinant or other means.

“Antibody fragment”, and all grammatical variants thereof, as usedherein is defined as a (1) portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains of the Fc regionof the intact antibody, and (2) constructs comprising a portion of anintact antibody (as defined by the amino acid sequence of the intactantibody) comprising the antigen binding site or variable region of theintact antibody.

An antibody fragment is, or comprises, a polypeptide having a primarystructure consisting of one uninterrupted sequence of contiguous aminoacid residues having the amino acid sequence of an intact antibody.Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂, Fd,Fc, Fv, diabodies, and any other “Non-single-chain antigen-binding unit”as described, e.g., in U.S. Pat. No. 7,429,652.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain connected to a light chain variable domain in the samepolypeptide chain. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, an “antigen-binding antibody fragment” is any antibodyfragment that retains the ability to bind to the specific target towhich the intact antibody specifically binds. An antigen-bindingantibody fragment may have different (e.g., lesser) binding affinity forthe target antigen than the intact antibody. As used herein, unlessotherwise stated, an antibody fragment is an antigen-binding antibodyfragment

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or, an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The terms “antigen”, “target molecule”, “target polypeptide”, “targetepitope”, and the like are used herein to denote the moleculespecifically bound by an antibody or antibody fragment.

As used herein, a “marker”, a “label”, a “marker moiety” and a “labelmoiety” refer to a detectable compound or composition which isconjugated directly or indirectly to the antibody so as to generate a“labeled” antibody. A label or marker provides a detectable signal forat least the time period during which a signal is to be observed. Thelabel or marker may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

A label or marker moiety may be, for example, a dye, an epitope tag, afluorescent moiety, a luminescent moiety, a chemiluminescent moiety, anenzymatic label, a magnetic label, a paramagnetic label, a contrastagent, a nanoparticle, a radioisotope, biotin, streptavidin, and aquencher. A nanoparticle may be a particle of an element, such as a goldnanoparticle, or of an alloy or compound, such as a quantum dot (aparticle of a semiconductor material), or other particle having a sizetypically in a range between about 1 nm to about 100 nm.

A label or marker moiety may provide a signal by reflecting, ormodulating, energy impinging on the label or marker moiety. A label ormarker moiety may provide a signal by emitting, or by increasing, adetectable signal. Similarly, a label or marker moiety may provide asignal by diminishing, or extinguishing, a signal (e.g., the quenchingof a signal). It will be understood that a label or marker moiety may bedirectly detectable (e.g., may provide a detectable signal withoutfurther action or input of energy), or may use or require energy, asubstrate, a binding partner, or other action in order to provide adetectable signal. An enzymatic label may be suitable for use with abinding partner or substrate; for example, a peroxidase such ashorseradish peroxidase may serve as a label as it may be used to detectthe presence of a target, or to measure the amount of target, when usedwith, e.g., diaminobenzidine or other molecule suitable for use with aperoxidase; for another, non-limiting example, luciferase may serve as alabel as it may be used to detect the presence of a target, or tomeasure the amount of target, when used with luciferin.

As used herein, the term “chromogen” refers to a compound which may bereadily converted into a dye or other colored compound.

As used herein, “BSA” means bovine serum albumin; “PEG” meanspolyethylene glycol; “ELISA” means enzyme-linked immunosorbent assay;and other terms, abbreviations, and acronyms have the standard meaningsunderstood in the chemical and biological arts.

A composition may include a buffer. Buffers include, without limitation,phosphate, citrate, ammonium, acetate, carbonate,tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino) propanesulfonicacid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO),2-(N-morpholino)ethanesulfonic acid (MES), N-(2-Acetamido)-iminodiaceticacid (ADA), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine chloride,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES),acetamidoglycine, tricine(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), glycinamide, andbicine (2-(Bis(2-hydroxyethyl)amino)acetic acid) buffers. Buffersinclude other organic acid buffers in addition to the phosphate,citrate, ammonium, acetate, and carbonate buffers explicitly mentionedherein.

A composition may include a physiologically acceptable carrier. Forexample, a physiologically acceptable carrier may be an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude buffers such as phosphate, citrate, and other organic acids asdiscussed above; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such as,e.g., serum albumin, gelatin, cytochromes, and immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, polysaccharides, and other carbohydrates includingglucose, mannose, and dextrins; chelating agents such as ethylenediamine tetraacetic acid (EDTA); sugar alcohols such as mannitol orsorbitol; salt-forming counterions such as sodium, potassium, calcium,magnesium and others; nonionic surfactants such as TWEEN™, polyethyleneglycol (PEG), and PLURONICS™; and/or other compounds known in the art.

For example, a composition may include albumin, gelatin, cytochrome C,an immunoglobulin, an amino acid, agar, glycerol, ethylene glycol, aprotease inhibitor, an antimicrobial agent, a metal chelating agent, amonosaccharide, a disaccharide, a polysaccharide, a reducing agent, achelating agent, or combinations thereof.

As used herein, a “sample”, or “biological sample”, or “clinical sample”refers to a sample of fluid, tissue, secretion, or excretion obtainedfrom a subject. A sample, biological sample, or clinical sample may be asample of blood, serum, plasma, saliva, sputum, urine, gastric fluid,digestive fluid, tears, sweat, stool, semen, vaginal fluid, interstitialfluid, fluid derived from tumorous tissue, ocular fluids, mucus, earwax,oil, glandular secretions, spinal fluid, skin, cerebrospinal fluid fromwithin the skull, tissue, fluid or material from a nasal swab, a throatswab, a mouth swab (e.g., a cheek swab), a vaginal swab, ornasopharyngeal wash, biopsy fluid or material, placental fluid, amnioticfluid, cord blood, lymphatic fluids, cavity fluids, pus, microbiotaobtained from a subject, meconium, breast milk, or other secretion orexcretion. A sample may be a breath sample, a hair sample, a fingernailsample, or other sample.

Biological and clinical samples may include nasopharyngeal wash, orother fluid obtained by washing a body cavity or surface of a subject,or by washing a swab following application of the swab to a body cavityor surface of a subject. Nasal swabs, mouth swabs (including cheekswabs), throat swabs, vaginal swabs, stool samples, hair, finger nail,ear wax, breath, and other solid, semi-solid, or gaseous samples may beprocessed in an extraction buffer, e.g., for a fixed or variable amountof time, prior to their analysis. The extraction buffer or an aliquotthereof may then be processed similarly to other fluid samples ifdesired. Examples of tissue samples of the subject may include but arenot limited to, connective tissue, muscle tissue, nervous tissue,epithelial tissue, cartilage, cancerous sample, or bone. The sample maybe obtained from a human or animal. The sample may be obtained from avertebrate, e.g., a bird, fish, or mammal, such as a rat, a mouse, apig, an ape, another primate (including humans), a farm animal, a sportanimal, or a pet. The sample may be obtained from a living or deadsubject. The sample may be obtained fresh from a subject or may haveundergone some form of pre-processing, storage, or transport.

As used herein, a “small volume” refers to a volume of less than about 1mL, or less than about 500 μL, or less than about 250 μL, or less than150 μL, or less than about 100 μL, or less than about 50 μL, or lessthan about 25 μL, or less. In particular embodiments, a small volume,such as a “finger-stick” volume, may comprise less than about 250 μL,and typically comprises less than 150 μL, or less than about 100 μL, orless than about 50 μL, or less than about 25 μL, or less.

A sample may be divided into two or more portions. As used herein, whenreferring to a sample or samples, the terms “portion” and “aliquot” andtheir plurals and grammatical equivalents are used interchangeably torefer to a fractional amount of sample taken from an original completesample. Such a fraction may be any fraction or amount, so that a portionor aliquot may comprise most of the original sample, a large fraction ofthe original sample, a small fraction of the original sample, or arelatively small fraction of the original sample. The phrases “at leasta portion”, “at least an aliquot”, and the like, may refer both to afractional part of an original sample and to the entire original sample.

Detection of markers, and detection of disease-causing (or other)organisms may include detection of nucleic acid markers; detection ofprotein (peptide) markers, including detection of antibodies; detectionof markers of inflammation (including both peptide and non-peptidemarkers); and detection of other markers. Identification of markers, andof disease-causing (or other) organisms may include identification ofnucleic acid markers; identification of protein (peptide) markers,including identification of antibodies; identification of markers ofinflammation (including both peptide and non-peptide markers); andidentification of other markers. Detection and identification of markersand organisms may include quantitative detection and identification ofsuch markers and such organisms.

A method may be performed in a short period of time. A device may becapable of performing all steps of a method in a short period of time. Adevice may be capable of performing all steps of a method on a singlesample in a short amount of time. A device may be capable of performingall steps of a method on two samples, such as a blood sample and asample obtained from a swab, in a short amount of time. A device may becapable of performing all steps of a method on more than two samples ina short amount of time. For example, from sample collection from asubject to detecting a disease marker, or to detecting multiple diseasemarkers, may take about 3 hours or less, 2 hours or less, 1 hour orless, 50 minutes or less, 45 minutes or less, 40 minutes or less, 30minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes orless, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutesor less, or 1 minute or less. For example, from sample collection from asubject to transmitting data regarding, and/or to analysis of, a sampleor samples may take about 3 hours or less, 2 hours or less, 1 hour orless, 50 minutes or less, 45 minutes or less, 40 minutes or less, 30minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes orless, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutesor less, or 1 minute or less.

For example, the period of time from initiating a method of detecting adisease marker to detecting a disease marker, or to detecting multipledisease markers, may be about 3 hours or less, 2 hours or less, 1 houror less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 30minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes orless, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutesor less, or 1 minute or less. For example, the period of time frominitiating a method of detecting a disease marker to transmitting dataregarding such detection may be about 3 hours or less, 2 hours or less,1 hour or less, 50 minutes or less, 45 minutes or less, 40 minutes orless, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes orless, 2 minutes or less, or 1 minute or less.

The period of time from accepting a sample within the device todetecting a disease marker, or to detecting a plurality of diseasemarkers, or to transmitting data regarding, and/or to analysis of, asample or samples may depend on the type or number of steps, tests, orassays performed on the sample or samples. The amount of time fromaccepting a sample, or samples, within the device to detecting a diseasemarker or markers, or to transmitting data and/or to analysis from thedevice regarding such a sample or samples may take about 3 hours orless, 2 hours or less, 1 hour or less, 50 minutes or less, 45 minutes orless, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes orless, 3 minutes or less, 2 minutes or less, or 1 minute or less.

Thus, as used herein, a “short period of time” refers to a period oftime of about 5 hours or less, or about 4 hours or less, or about 3hours or less, or about 2 hours or less, or about 1 hour or less, orabout 50 minutes or less, or about 40 minutes or less, or about 30minutes or less, or about 20 minutes or less, or about 10 minutes orless, or about 5 minutes or less. A short period of time may bedetermined with respect to an initial time; the initial time may be thetime at which a sample analysis began; the initial time may be the timeat which a sample is inserted into a device for the analysis of thesample; the initial time may be the time at which a sample was obtainedfrom a subject.

The terms “point of service” (abbreviated POS) and “point of servicesystem,” as used herein, refer to a location, and a system at thatlocation, that is capable of providing a service (e.g. testing,monitoring, treatment, diagnosis, guidance, sample collection,verification of identity (ID verification), and other services) at ornear the site or location of the subject. A service may be a medicalservice, and may be a non-medical service. In some situations, a POSsystem provides a service at a predetermined location, such as asubject's home, school, or work, or at a grocery store, a drug store, acommunity center, a clinic, a doctor's office, a hospital, etc. A POSsystem can include one or more point of service devices. In someembodiments, a POS system is a point of care system.

A “point of care” (abbreviated POC) is a location at whichmedical-related care (e.g. treatment, testing, monitoring, diagnosis,counseling, etc.) is provided. A POC may be, e.g. at a subject's home,work, or school, or at a grocery store, a community center, a drugstore, a doctor's office, a clinic, a hospital, etc. A POC system is asystem which may aid in, or may be used in, providing suchmedical-related care, and may be located at or near the site or locationof the subject or the subject's health care provider (e.g. subject'shome, work, or school, or at a grocery store, a community center, a drugstore, a doctor's office, a clinic, a hospital, etc.).

As used herein, the term “immunoassay” refers to any assay whichdetects, identifies, characterizes, quantifies, or otherwise measures anamino acid target in a sample (where an amino acid target may be a smallpeptide, a polypeptide, a protein, or proteinaceous macromolecule).Immunoassays include, for example, direct or competitive binding assaysusing techniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, fluorescent immunoassays, and protein Aimmunoassays. Immunoassays typically use antibodies or antibodyfragments, but may also use binding proteins or carrier proteins whichbind target molecules with high specificity.

As used herein, the term “nucleic acid assay” is used to refer to anyassay which detects, identifies, characterizes, quantifies, or otherwisemeasures a nucleic acid target in a sample (where a nucleic acid targetmay be a single stranded, double stranded, or other nucleic acidmolecule of any size. Nucleic acid assays include polymerase chainreaction (PCR) assays (see, e.g. U.S. Pat. No. 4,683,202), loop-mediatedisothermal amplification (“LAMP”) (see, e.g. U.S. Pat. No. 6,410,278),and other methods, including methods discussed below for detectingnucleic acid targets in a sample. Nucleic acid markers may be detectedby any suitable means, including means that include nucleic acidamplification (e.g., thermal cycling amplification methods includingPCR, and other nucleic acid amplification methods; isothermalamplification methods, including LAMP, etc.) and any other method thatcan be used to detect the presence of nucleic acid markers indicative ofa disease-causing organism in a sample.

As used herein, the term “general chemistry assay” refers to any assaywhich detects, identifies, characterizes, quantifies, or otherwisemeasures a target in a sample, other than a target which is a nucleicacid or other than by use of an antibody or other specifically bindingprotein. General chemistry assays include, e.g., assays forelectrolytes; for vitamin levels; for blood component levels; for tracemetals; for lipids; and other targets). General chemistry assays mayinclude, for example, assays of a Basic Metabolic Panel [glucose,calcium, sodium (Na), potassium (K), chloride (Cl), CO₂ (carbon dioxide,bicarbonate), creatinine, blood urea nitrogen (BUN)], assays of anElectrolyte Panel [sodium (Na), potassium (K), chloride (Cl), CO₂(carbon dioxide, bicarbonate)], assays of a Chem 14 Panel/ComprehensiveMetabolic Panel [glucose, calcium, albumin, total protein, sodium (Na),potassium (K), chloride (Cl), CO₂ (carbon dioxide, bicarbonate),creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP),alanine aminotransferase (ALT/GPT), aspartate aminotransferase(AST/GOT), total bilirubin] assays of a Lipid Profile/Lipid Panel [LDLcholesterol, HDL cholesterol, total cholesterol, and triglycerides],assays of a Liver Panel/Liver Function [alkaline phosphatase (ALP),alanine aminotransferase (ALT/GPT), aspartate aminotransferase(AST/GOT), total bilirubin, albumin, total protein, gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), prothrombin time (PT)],alkaline phosphatase (APase), hemoglobin, VLDL cholesterol, ethanol,lipase, pH, zinc protoporphyrin, direct bilirubin, blood typing (ABO,RHD), lead, phosphate, hemagglutination inhibition, magnesium, iron,iron uptake, fecal occult blood, and others, individually or in anycombination.

As used herein the term “cytometric assay” refers to any which detects,identifies, characterizes, quantifies, or otherwise measures a cell orlarge particle (e.g., a crystal) in a sample. Cytometric assaystypically utilize imaging of other light-based techniques to detect,measure, characterize, and quantify cells and particles in a sample.

Systems, Devices, and Methods

In embodiments, Applicants disclose systems, including such systemsdiscussed above, for detecting one or more of a plurality ofdisease-causing agents in a clinical sample. In embodiments, Applicantsdisclose systems, including such systems discussed above, for detectingone or more of a plurality of disease-causing agents in a clinicalsample, wherein said diseases comprise respiratory diseases. Inembodiments, Applicants disclose systems, including such systemsdiscussed above, for detecting one or more of a plurality ofdisease-causing agents in a clinical sample, wherein saiddisease-causing agents cause respiratory diseases selected from viraldiseases, bacterial diseases, fungal diseases, mycoplasma diseases, andother diseases.

In embodiments, the plurality of disease-causing agents causes a numberof diseases, wherein said number of diseases comprises 8 or morediseases, or 10 or more diseases, or 12 or more diseases, or 14 or morediseases, or 16 or more diseases, or 18 or more diseases, or 20 or morediseases, or 30 or more diseases, or 40 or more diseases, or 50 or morediseases, or 60 or more diseases, or more. In embodiments, the pluralityof disease-causing agents causes a number of diseases, wherein saiddiseases are selected from viral diseases, bacterial diseases, fungaldiseases, mycoplasma diseases, and other diseases.

In embodiments, the systems disclosed herein, and the methods disclosedherein, may be used to perform all of a plurality of assays on a singlesmall volume sample, or an aliquot or aliquots thereof. In embodiments,a small volume sample has a volume selected from no more than about 1mL, or no more than about 500 μL, or no more than about 250 μL, or nomore than about 150 μL, or no more than about 100 μL, or no more thanabout 75 μL, or no more than about 50 μL, or no more than about 25 μL,or no more than about 15 μL, or no more than about 10 μL, or no morethan about 5 μL, or no more than about 4 μL, or no more than about 3 μL,or no more than about 2 μL, or no more than about 1 μL, or less thanabout 1 μL.

In embodiments, the systems disclosed herein, and the methods disclosedherein, may be used to perform all of a plurality of assays in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about 2 hours, or less than about 1hour, or less than about 50 minutes, or less than about 45 minutes, orless than about 40 minutes, or less than about 30 minutes, or less thanabout 20 minutes, or less than about 15 minutes, or less than about 10minutes, or less than about 5 minutes, or less than about 4 minutes, orless than about 3 minutes, or less than about 2 minutes, or less thanabout 1 minute.

In embodiments, the plurality of disease-causing agents may cause anumber of diseases, wherein said disease-causing agents are selectedfrom mycobacterium tuberculosis, staphylococcus aureus (includingmethicillin-resistant staphylococcus aureus), streptococcus (includingstreptococcus Group A and streptococcus Group B), bordetella pertussis,adenovirus (including adenovirus B, adenovirus C, and adenovirus E),influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus,corona virus, bocavirus, haemophilus parainfluenzae, human papillomavirus (HPV), hepatitis, human immunodeficiency virus (HIV), herpessimplex virus (HSV), West Nile, Epstein Barr, Rhinovirus, and otherviruses. In embodiments, the plurality of disease-causing agents causesa number of diseases, wherein said disease-causing agents are selectedfrom streptococcus, staphylococcus, bordetella pertussis, tuberculosis,enterobacteria, pseudomonas, dengue, malaria, trypanosome cruzi,treponema pallidum, mycoplasma, chlamydia, Moraxella catarrhalis,acinetobacter, legionella, Escherichia coli, candida, chlamydia,Neisseria, trichomonas, and other micro-organismal disease-causingagents, including but not limited to disease-causing agents namedelsewhere herein.

In embodiments, the plurality of disease-causing agents may cause anumber of respiratory diseases, wherein said number of respiratorydiseases comprises 8 or more respiratory diseases, or 10 or morerespiratory diseases, or 12 or more respiratory diseases, or 14 or morerespiratory diseases, or 16 or more respiratory diseases, or 18 or morerespiratory diseases, or 20 or more respiratory diseases, or 30 or morerespiratory diseases, or 40 or more respiratory diseases, or 50 or morerespiratory diseases, or 60 or more respiratory diseases, or more.

In embodiments, the plurality of disease-causing agents may cause anumber of respiratory diseases, wherein said respiratory diseases areselected from viral diseases, bacterial diseases, fungal diseases,mycoplasma diseases, and other diseases.

In embodiments, the plurality of disease-causing agents may cause anumber of respiratory diseases, wherein said respiratory disease-causingagents are selected from mycobacterium tuberculosis, staphylococcusaureus (including methicillin-resistant staphylococcus aureus),streptococcus (including streptococcus Group A), bordetella pertussis,adenovirus (including adenovirus B, adenovirus C, and adenovirus E). Inembodiments, respiratory disease-causing agents may further include oneor more of influenza, parainfluenza, respiratory syncytial virus (RSV),adenovirus, corona virus, bocavirus, haemophilus parainfluenzae, humanpapilloma virus (HPV), hepatitis, human immunodeficiency virus (HIV),herpes simplex virus (HSV), West Nile, Epstein Barr, Rhinovirus, andother viruses.

In embodiments, the plurality of disease-causing agents may causeinfluenza. In embodiments, the influenza may be selected from influenzaA, influenza B, H1N1 influenza (including seasonal and novel forms ofinfluenza H1N1), H3N2 influenza, H7N9 influenza, H5N1 influenza, andother influenzas.

In embodiments, the plurality of disease-causing agents may cause asexually transmitted disease. In embodiments, the sexually transmitteddisease is selected from herpes simplex virus (HSV), humanimmunodeficiency virus (HIV, including HIV-1, HIV-2, including HIV-2Group A), gonnorhea, syphilis, human papilloma virus (PPV),streptococcus (including streptococcus B), treponema pallidum, and othersexually transmitted diseases.

Further targets include drug-resistant micro-organisms, including thoseexhibiting multi-drug resistance. Drug resistance (also termedantibiotic resistance) is found where a population (or subpopulation) ofa micro-organism, such as a bacterium, acquires or exhibits resistanceto one or more drugs (e.g., to one or more antibiotic). Micro-organismsthat are resistant to treatment by multiple drugs are termed to be“multi-drug resistant” and those micro-organisms are termed to have orto exhibit “multi-drug resistance”; either term may be abbreviated by“MDR”. Resistance to one or more drugs is observed, or exhibited, when apopulation of micro-organisms survives (and typically continues to growand multiply in number) despite the presence of a drug, or (in the caseof MDR) despite the presence of multiple drugs. Drug-resistant organismsof particular interest include, but are not limited to,Methicillin-Resistant Staphylococcus aureus (MRSA),vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus(VRSA), bacteria (e.g., Enterobacteriaceae) having extended spectrumbeta-lactamase (ESBL), Vancomycin-resistant Enterococcus (VRE), andMultidrug-resistant A. baumannii (MRAB). Drug-resistant targetorganisms, including MDR target organisms, may be identified by nucleicacid markers, protein (or peptide) markers, other markers, orcombinations thereof, as well as by observing their growth in thepresence of drugs.

Many antibiotic compounds include a β-lactam ring (a ring of fourcarbons); for example, penicillin is an antibiotic having a β-lactamring. Many bacteria have β-lactamase enzymes which can cleave a β-lactamring, and thus protect the bacteria against such antibiotics. Enzymesthat can cleave a β-lactam ring, which are often found in Gram-negativebacteria, include the TEM and ROB β-lactamase enzymes. Drug-resistantHaemophilus influenzae bacteria may have the blaTEM or blaROB resistancegene (typically blaTEM-1, although blaTEM-2 and blaROB-1 and others arealso found); other drug-resistance markers found in disease-causingorganisms include the KPC resistance gene (found in Klebsiella pneumoniacarbapenemas (KPC)); mecA and mecC resistance genes (responsible forresistance to β-lactam-containing antibiotics such as methicillin);vancomycin resistance genes A and B (vanA and vanB) may be found indisease-causing bacteria, such as, e.g., vancomycin resistantEnterococci; and others drug-resistance markers.

Disease-causing organisms of interest include viruses of the filoviridaefamily of viruses (filo viruses), which includes Ebola viruses, Marburgviruses, and Cueva viruses. Ebola viruses cause ebola virus disease(also known as Ebola hemorrhagic fever); Marburg viruses also cause ahemorrhagic fever, the Marburg hemorrhagic fever; and Cueva viruses suchas lloviu virus (LLOV), may be endemic in France, Spain, or Portugal.Such viruses may be detected and may be identified in a sample fromnucleic acid markers specific to these viruses, from protein (peptide)markers specific to these viruses, and from other markers specific tothese viruses.

Filo viruses typically cause hemorrhagic fevers and related disorders.These and other diseases may be detected, and may be identified, byidentifying nucleic acid markers, peptide markers, and other markers,alone or in combination, as disclosed herein. Filo viral diseases, otherhemorrhagic fevers, and other diseases including many tropical diseases,e.g. Dengue 1, Dengue 2, Dengue 3, Dengue 4, malaria, typhoid, and otherdiseases, have many detrimental effects, including hemorrhage andinternal bleeding, and may cause disruptions in electrolytes, may causeanemia, and may cause other symptoms and effects. Integrated electrolyteassays (e.g., for sodium, potassium, and other electrolytes, includingsodium and potassium together, and including sodium and potassiumtogether with other electrolytes) may identify subjects suffering fromelectrolyte imbalances, and may thus identify subjects suffering from ahemorrhagic fever, from anemia, or both. Assays for hemoglobin, foriron, and other assays may identify subjects suffering from anemia, froma hemorrhagic fever, or both. Anemia may be due to hemorrhage, parasiticinfection (e.g., hookworm), both hemorrhage and parasitic infection, andother causes. Thus, in addition to testing for nucleic acid, peptide,and other markers, these hemorrhagic and other diseases may be detected,and may be identified, with integrated electrolyte measurements; withhemoglobin measurements; with iron measurements; and with other generalchemistry assays, alone or in combination. In embodiments, integratedelectrolyte measurements, hemoglobin, iron, or other general chemistryassays may provide indications that a subject suffers from anemia, ahemorrhagic fever such as Ebola, Marburg, or other disease such asmalaria or typhoid fever; such indication may be used to suggest furthertesting for markers of such diseases. Thus, in embodiments, electrolytetesting including sodium and potassium, or other general chemistryassays, may be performed simultaneously when testing for Ebola, Marburg,or other hemorrhagic disease (e.g., when testing for nucleic acid,peptide, or other markers for such diseases).

In embodiments, such indications derived from general chemistry testresults (e.g., from the results of integrated electrolyte measurements,hemoglobin, iron, or other general chemistry assays) may automaticallytrigger reflex testing for one or more hemorrhagic fever diseasemarkers, or malaria, typhoid fever, or other disease marker, and mayautomatically trigger reflex testing for combinations or for all of suchmarkers. In embodiments, a subject may be tested for other diseases andnot (initially) for Ebola, Marburg, or other hemorrhagic disease (e.g.,a patient that is weak, or feverish, may not initially be tested forEbola, Marburg, or other hemorrhagic disease); based on the results ofintegrated electrolyte measurements, hemoglobin, iron, or other generalchemistry assays, a reflex test for Ebola, Marburg, or other hemorrhagicdisease may be automatically performed, or may be ordered by a healthcare professional. In embodiments, a patient or subject may not wish tobe tested for Ebola, Marburg, or other hemorrhagic disease, or otherdisease of concern (e.g., a patient may fear quarantine, or may fearthat the cost of the test might be prohibitive) and thus may notinitially be tested for Ebola, Marburg, or other hemorrhagic disease;based on the results of integrated electrolyte measurements, hemoglobin,iron, or other general chemistry assays, a reflex test for Ebola,Marburg, or other hemorrhagic disease may be automatically performed, ormay be ordered by a health care professional. In embodiments, such areflex test for Ebola, Marburg, or other hemorrhagic disease, or otherdisease of concern may be performed if the patient is first tested forone or more infectious disease other than Ebola, Marburg, or otherhemorrhagic disease, and the initial test panel does not show infectionof any known disease (and thus further testing would be required inorder to identify other possible sources of the subject's disease orcondition).

Methods, devices, and systems disclosed herein may be used to detect,and may be used to identify, disease-causing organisms in normal orhealthy individuals and populations. Methods, devices, and systemsdisclosed herein may be used to detect, and may be used to identify,benign organisms in normal or healthy individuals and populations. Suchdisease-causing organisms and such benign organisms may be detected, andmay be identified, in samples obtained from a normal individual, e.g.,once or on an on-going basis, in order to determine a baseline or normallevel for that individual when that individual is healthy. Suchdetection of disease-causing and benign organisms may include detectionof nucleic acid markers; detection of protein (peptide) markers,including detection of antibodies; detection of markers of inflammation(including both peptide and non-peptide markers); and detection of othermarkers. Such identification of disease-causing and benign organisms mayinclude identification of nucleic acid markers; identification ofprotein (peptide) markers, including identification of antibodies;identification of markers of inflammation (including both peptide andnon-peptide markers); and identification of other markers. Detection andidentification of markers may include quantitative detection andidentification of such markers. Differences between the results from asample and normal or baseline results may be used to improve thelikelihood of detecting whether or not an individual suffers from adisease or condition. For example, determination of a baseline or normallevel for an individual aids in detecting, in identifying, and indiagnosing disease conditions, or progression towards a disease or adetrimental condition, by comparison of results from later-obtainedsamples to baseline or normal levels determined when the individual ashealthy. Such comparisons between an individual subject's results andbaseline or normal levels found in prior results obtained from theindividual when healthy can be used to determine if it is likely thatthe individual subject suffers from an infection. Such comparisons mayinclude consideration of symptoms, if any, of that individual subject incomparison to symptoms (or the lack thereof) previously found for thatindividual when healthy.

Such disease-causing organisms and such benign organisms may be may bedetected, and may be identified, in samples obtained from multipleindividuals, e.g., once or on an on-going basis, in order to determine abaseline or normal level found in a normal (healthy) population. Suchdetection of disease-causing and benign organisms may include detectionof nucleic acid markers; detection of protein (peptide) markers,including detection of antibodies; detection of markers of inflammation(including both peptide and non-peptide markers); and detection of othermarkers. Such identification of disease-causing and benign organisms mayinclude identification of nucleic acid markers; identification ofprotein (peptide) markers, including identification of antibodies;identification of markers of inflammation (including both peptide andnon-peptide markers); and identification of other markers. Detection andidentification of markers may include quantitative detection andidentification of such marker. Differences between the results obtainedfrom a sample from an individual subject and normal or baseline resultsobtained from a population of comparable healthy individuals may be usedto improve the likelihood of detecting whether or not an individualsubject suffers from a disease or condition. For example, determinationof a baseline or normal level for an individual subject aids indetecting, in identifying, and in diagnosing disease conditions, orprogression towards a disease or a detrimental condition, by comparisonof results for the individual subject to baseline or normal levels foundin a population of comparable healthy individuals. Such comparisonsbetween an individual subject's results and baseline or normal levelsfound in a healthy population can be used to determine if it is likelythat the individual subject suffers from an infection. Such comparisonsmay include consideration of symptoms, if any, of that individualsubject in comparison to symptoms (or the lack thereof) found in ahealthy population.

In embodiments, such a system is a point-of-service system (POS system),wherein a POS system is located at a point of service location. Inembodiments, a POS system is located at a point of service location andis configured to accept a clinical sample obtained from a subject at thePOS location. In embodiments, a POS system is located at a point ofservice location and is configured to accept a clinical sample obtainedfrom a subject at the POS location, and is further configured to analyzethe clinical sample at the POS location. In embodiments, the clinicalsample is a small volume clinical sample. In embodiments, the clinicalsample is analyzed in a short period of time. In embodiments, the shortperiod of time is determined with respect to the time at which sampleanalysis began. In embodiments, the short period of time is determinedwith respect to the time at which the sample was inserted into a devicefor the analysis of the sample. In embodiments, the short period of timeis determined with respect to the time at which the sample was obtainedfrom the subject.

Applicants disclose herein methods for detecting the presence of atarget disease-causing agent, or marker indicative of the presence of atarget disease-causing agent, in a single small-volume sample or aliquotthereof. In embodiments, methods for detecting the presence of aplurality of target disease-causing agents, or markers indicativethereof, from a single sample, or aliquot thereof, within a short periodof time are disclosed. In embodiments, the plurality of targetdisease-causing agents, or markers indicative thereof, termed “targets”,comprises at least 5 targets, or at least 10 targets, or at least 15targets, or at least 20 targets, or at least 25 targets, or at least 30targets, or at least 35 targets, or at least 40 targets, or at least 45targets, or at least 50 targets, or at least 55 targets, or at least 60targets, or at least 65 targets, or more. In embodiments, a short periodof time is a period of time that is five hours or less, or is four hoursor less, or is three hours or less, or is two hours or less, or is onehour or less, or is 50 minutes or less, or is 40 minutes or less, or is30 minutes or less, or is 20 minutes or less, or is 10 minutes or less,or is 5 minutes or less.

Applicants disclose herein methods for detecting the presence of atarget flu virus molecule in a sample are disclosed herein, wherein thepresence of a plurality of possible target flu viruses are tested from asingle sample within a short period of time. In embodiments, theplurality of possible target flu viruses comprise at least 5 possibletarget flu viruses, or at least 10 possible target flu viruses, or atleast 15 possible target flu viruses, or at least 20 possible target fluviruses, or at least 25 possible target flu viruses, or at least 30possible target flu viruses, or at least 35 possible target flu viruses,or at least 40 possible target flu viruses, or at least 45 possibletarget flu viruses, or at least 50 possible target flu viruses, or atleast 55 possible target flu viruses, or at least 60 possible target fluviruses, or at least 64 possible target flu viruses, or at least 65possible target flu viruses, or more.

Applicants further disclose herein methods for detecting the presence ofa plurality of target molecules in a single sample within a short periodof time, wherein the plurality of target molecules comprises nucleicacid molecules, or protein molecules, or saccharides, or cytokines, orsteroids, or histamine, or other molecules. Applicants further discloseherein methods for detecting the presence of a plurality of targetmolecules in a single sample within a short period of time, wherein theplurality of target molecules comprises nucleic acid molecules andprotein molecules. Applicants further disclose herein methods fordetecting the presence of a plurality of target molecules in a singlesample within a short period of time, wherein the plurality of targetmolecules comprises nucleic acid molecules, protein molecules, markersof inflammation, and cytokines. Applicants further disclose hereinmethods for detecting the presence of a plurality of target molecules ina single sample within a short period of time, wherein the plurality oftarget molecules comprises nucleic acid molecules, protein molecules,and saccharides.

Applicants disclose herein devices for use in systems and methods asdisclosed herein, Such devices include, for example, devices comprisinga holder configured to accept and retain a clinical sample (e.g., aclinical sample contained within a sample collection device); a reagentvessel or a plurality of reagent vessels; and a reaction vessel, or aplurality of reaction vessels. In embodiments, such devices may furtherconfigured to accept and retain one or more of a cytometry cuvette orcuvettes; a waste container or containers; a tip, or tips, configured toaspirate or release fluid; and other tools.

Applicants further disclose herein assays for the detection of one ormore of a plurality of target molecules in a single sample within ashort period of time, wherein the plurality of target moleculescomprises one or more of nucleic acid molecules, protein molecules,saccharides, markers of inflammation, and cytokines. In embodiments,such assays may be configured for use with systems and devices asdisclosed herein.

Accordingly, Applicants disclose herein systems, devices, and methods,including the following exemplary integrated systems.

1) An integrated system for providing testing and diagnosis of a subjectsuspected of suffering from a disease, said system comprising a meansfor obtaining a sample (which may include, e.g., a sample collectiondevice comprising a lancet, a syringe, a needle and tube, or other bloodcollection device; or a nasal swab, a mouth swab (e.g., a cheek swab), athroat swab, a vaginal swab, or other swab, and fluid in which toimmerse the swab following contacting the swab with a subject); acartridge comprising reagents for assays for the disease; a device forrunning a plurality of assays for detecting a plurality of diseases; adevice/means for displaying/communicating the detection of one or moreof said diseases. Such integrated systems may be configured for useswherein the sample is a small volume sample; for uses wherein detectionis performed in a short period of time; or for uses both wherein thesample is a small volume sample and wherein detection is performed in ashort period of time.

2) An integrated system for providing testing and diagnosis of a subjectsuspected of suffering from a respiratory disorder, said systemcomprising a means for obtaining a sample (which may include, e.g., anasal swab, a throat swab, a mouth swab (e.g., a cheek swab), a vaginalswab, or other swab, and fluid in which to immerse the swab followingcontacting the swab with a subject); a cartridge comprising reagents forassays for respiratory disorders; a device for running a plurality ofassays for detecting a plurality of respiratory disorders; adevice/means for displaying/communicating the detection of one or moreof said respiratory disorders. Such integrated systems may be configuredfor uses wherein the sample is a small volume sample; for uses whereindetection is performed in a short period of time; or for uses bothwherein the sample is a small volume sample and wherein detection isperformed in a short period of time.

3) An integrated system for providing testing, diagnosis, andprescription of a subject suspected of suffering from a respiratorydisorder, said system comprising a means for obtaining a sample (whichmay include, e.g., a nasal swab, a throat swab, a mouth swab (e.g., acheek swab), a vaginal swab, or other swab, and fluid in which toimmerse the swab following contacting the swab with a subject); acartridge comprising reagents for assays for respiratory disorders; adevice for running a plurality of assays for detecting a plurality ofrespiratory disorders; a device/means for displaying/communicating thedetection of one or more of said respiratory disorders; and means forproviding a prescription for the treatment of a respiratory disorderdetected in said sample. Such integrated systems may be configured foruses wherein the sample is a small volume sample; for uses whereindetection is performed in a short period of time; or for uses bothwherein the sample is a small volume sample and wherein detection isperformed in a short period of time.

4) An integrated system for providing testing, diagnosis, prescription,and treatment of a subject suspected of suffering from a respiratorydisorder, said system comprising a means for obtaining a sample (whichmay include, e.g., a nasal swab, a throat swab, a mouth swab (e.g., acheek swab), a vaginal swab, or other swab, and fluid in which toimmerse the swab following contacting the swab with a subject); acartridge comprising reagents for assays for respiratory disorders; adevice for running a plurality of assays for detecting a plurality ofrespiratory disorders; a device/means for displaying/communicating thedetection of one or more of said respiratory disorders; means forproviding a prescription for the treatment of a respiratory disorderdetected in said sample; and means for providing/selling/delivering atreatment (drug/pill/shot) to said subject pursuant to saidprescription. Such integrated systems may be configured for uses whereinthe sample is a small volume sample; for uses wherein detection isperformed in a short period of time; or for uses both wherein the sampleis a small volume sample and wherein detection is performed in a shortperiod of time.

Methods of detecting the presence of a target flu virus molecule in asample are disclosed herein, wherein the presence of a plurality ofpossible target flu viruses are tested from a single sample within ashort period of time. In embodiments, the plurality of possible targetflu viruses comprise at least 5 possible target flu viruses, or at least10 possible target flu viruses, or at least 15 possible target fluviruses, or at least 20 possible target flu viruses, or at least 25possible target flu viruses, or at least 30 possible target flu viruses,or at least 35 possible target flu viruses, or at least 40 possibletarget flu viruses, or at least 45 possible target flu viruses, or atleast 50 possible target flu viruses, or at least 55 possible target fluviruses, or at least 60 possible target flu viruses, or at least 64possible target flu viruses, or at least 65 possible target flu viruses,or more. In embodiments, a short period of time is a period of time thatis five hours or less, or is four hours or less, or is three hours orless, or is two hours or less, or is one hour or less, or is 50 minutesor less, or is 40 minutes or less, or is 30 minutes or less, or is 20minutes or less, or is 10 minutes or less, or is 5 minutes or less.

Methods of detecting the presence of a respiratory disease-causing agentin a subject suspected of having a respiratory disorder, wherein thepresence of a plurality of possible respiratory disease-causing agentsare tested from a single sample using both nucleic acid testing andprotein testing, wherein nucleic acid testing comprises detection of thepresence of target nucleic acid sequences, and wherein protein testingcomprises detection of the presence of target proteins having targetamino acid sequences. In embodiments, target nucleic acid sequences maycomprise sequences having at least 8 nucleotides, or at least 10nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, orat least 30 nucleotides, or at least 40 nucleotides, or at least 50nucleotides, or more, that are identical, or closely similar, to targetnucleotide sequences. In embodiments, target amino acid sequences maycomprise sequences having at least 8 amino acids, or at least 10 aminoacids, or at least 15 amino acids, or at least 20 amino acids, or atleast 30 amino acids, or at least 40 amino acids, or at least 50 aminoacids, or more, that are identical, or closely similar, to target aminoacids sequences.

In embodiments, a respiratory disease-causing agent is detected if morethan a minimum level of such respiratory disease-causing agents isdetected in a small-volume sample obtained from a subject, wherein thesmall-volume sample is tested for the presence of a plurality ofrespiratory disease-causing agents. In embodiments, the small-volumesample is 150 μL or less in volume, or is 75 μL or less in volume, or is50 μL or less in volume, or is 25 μL or less in volume, or is 15 μL orless in volume, or is 10 μL or less in volume, or is 5 μL or less involume.

Embodiments of methods disclosed herein include methods in which adisease-causing agent is detected if more than a minimum level of suchdisease-causing agents is detected in a small-volume sample obtainedfrom a subject. For example, such methods include methods wherein asmall-volume sample is tested for the presence of a plurality ofdisease-causing agents. In embodiments of such methods, that minimumlevel may be set at a level that is determined by the condition of thesubject. The minimum level may be set at a higher level for subjects whoexhibit symptoms of active infection. The minimum level may be set at alower level for subjects who are receiving treatment for an infection atthe time the sample was obtained. The minimum level may be set at alower level for subjects who have received treatment for an infectionprior to the time the sample was obtained, e.g., who have recentlyreceived treatment for an infection prior to the time the sample wasobtained.

In embodiments, the sample may be diluted prior to testing for thepresence of a plurality of disease-causing agents. In embodiments, suchdilution of a sample is greater for subjects who have a condition whichindicates they may have higher levels of disease-causing agents thansubject who do not have that condition, or than subjects who have adifferent condition.

Embodiments of methods disclosed herein include methods in which arespiratory disease-causing agent is detected if more than a minimumlevel of such respiratory disease-causing agents is detected in asmall-volume sample obtained from a subject. For example, such methodsinclude methods wherein a small-volume sample is tested for the presenceof a plurality of respiratory disease-causing agents. In embodiments ofsuch methods, that minimum level may be set at a level that isdetermined by the condition of the subject. The minimum level may be setat a higher level for subjects who exhibit symptoms of active infection.The minimum level may be set at a lower level for subjects who arereceiving treatment for an infection at the time the sample wasobtained. The minimum level may be set at a lower level for subjects whohave received treatment for an infection prior to the time the samplewas obtained, e.g., who have recently received treatment for aninfection prior to the time the sample was obtained.

In embodiments, the sample may be diluted prior to testing for thepresence of a plurality of disease-causing agents, such as respiratorydisease-causing agents. In embodiments, such dilution of a sample isgreater for subjects who have a condition which indicates they may havehigher levels of disease-causing agents, such as respiratorydisease-causing agents, than subject who do not have that condition, orthan subjects who have a different condition.

For example, conditions which indicate that a subject may have higherlevels of disease-causing agents, such as respiratory disease-causingagents, include subjects with an active infection; subjects who have acough, including a persistent cough; subjects who have a fever; subjectswho report chills; subjects who report fatigue; subjects who report aheadache; subjects who have sweats; and subjects who have or reportother symptoms indicative of an active infection.

For example, conditions which indicate that a subject may not havehigher levels of disease-causing agents, such as respiratorydisease-causing agents, include subjects currently receiving treatmentfor infection; subjects who recently received treatment for infection;subjects currently receiving, or who recently received treatment for, acough, including a persistent cough; a fever; chills; fatigue; headache;sweats; or other symptoms or indication of an infection.

In embodiments of methods in which a disease-causing agents, such as arespiratory disease-causing agent, is detected if more than a minimumlevel of such disease-causing agent (e.g., a respiratory disease-causingagent) is detected in a small-volume sample obtained from a subject,wherein the small-volume sample is tested for the presence of aplurality of disease-causing agents, such as respiratory disease-causingagents, the minimum level is set at a higher level for subjects who havenot been recently diagnosed with a disease, such as a respiratorydisease, than the minimum level set for subjects who have been recentlydiagnosed with a disease (such as a respiratory disease). Inembodiments, the sample may be diluted prior to testing for the presenceof said plurality of disease-causing agents, such as respiratorydisease-causing agents. In embodiments, such dilution of a sample isgreater for subjects who have not been recently diagnosed with adisease, such as a respiratory disease, than the dilution of samplesobtained from subjects who have been recently diagnosed with a disease,such as a respiratory disease.

In embodiments, the sample may be further tested for the presence ofindicators of inflammation. For example, the sample may be furthertested for the presence of higher than normal levels of glucocorticoidssuch as cortisol (e.g., in the blood, or in saliva, or tears, or otherbodily fluid or sample obtained by a swab). For example, the sample maybe further tested for the presence of higher than normal levels ofhistamine (e.g., in the blood, or in saliva, or tears, or other bodilyfluid or sample obtained by a swab). Further indicators of inflammationinclude, without limitation, increased levels of prostaglandins,increased levels of inflammatory cytokines (including, e.g., tumornecrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interleukin-18 (IL-18), and interferongamma (IF-γ)), bradykinin, complement system molecules, blood-clottingfactors, C-reactive protein, erythrocyte sedimentation rate (ESR), whiteblood cell count, morphological changes in blood and other cells, andother molecular and cellular markers indicative of inflammation. Inaddition, a subject may be examined, or asked to self-report, symptomsof inflammation, such as, e.g., swelling, redness, pain, or sensation ofheat of an affected area or tissue.

In embodiments, the sample may be further tested for the presence of acytokine or of a plurality of cytokines. In embodiments, the sample maybe further tested for the level of a cytokine or of a plurality ofcytokines. In embodiments, the target cytokine is selected from alymphokine, a chemokine, an interleukin, and an interferon. Inembodiments, the target cytokines are selected from lymphokines,chemokines, interleukins, and interferons. In embodiments, the cytokinemay be an inflammatory cytokine. In embodiments, the cytokine may be ananti-inflammatory cytokine. In embodiments, a target cytokine may be aan interleukin (IL) selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, and other interleukins. Inembodiments, a target cytokine may be a chemokine selected from an achemokine (also termed a CXC chemokine), a β-chemokine (also termed aCC-chemokine), a γ-chemokine (also termed a C-chemokine), and ad-chemokine (also termed a CX₃C-chemokine). In embodiments, a targetcytokine may be a member of the tumor necrosis factor (TNF) family.

In embodiments, the same sample may be tested for disease-causing agentsand for cytokines. In embodiments, the same sample may be tested forrespiratory disease-causing agents and for cytokines.

In embodiments, the sample may be further tested for the presenceof/antibodies to a disease-causing agent. In embodiments, the samesample may be tested for a plurality of disease-causing agents, and forantibodies to a plurality of disease-causing agents.

In embodiments, the sample may be further tested for the presence ofantibodies to a respiratory disease-causing agent. In embodiments, thesame sample may be tested for a plurality of respiratory disease-causingagents and for antibodies to a plurality of respiratory disease-causingagents.

Methods of detecting the presence of a target flu virus molecule in asample are disclosed herein, wherein the presence of a plurality ofpossible target flu viruses are tested from a single sample using bothnucleic acid testing and protein testing, wherein nucleic acid testingcomprises detection of the presence of target nucleic acid sequences,and wherein protein testing comprises detection of the presence oftarget proteins having target amino acid sequences. In embodiments,target nucleic acid sequences may comprise sequences having at least 8nucleotides, or at least 10 nucleotides, or at least 15 nucleotides, orat least 20 nucleotides, or at least 30 nucleotides, or at least 40nucleotides, or at least 50 nucleotides, or more, that are identical, orclosely similar, to target nucleotide sequences. In embodiments, targetamino acid sequences may comprise sequences having at least 8 aminoacids, or at least 10 amino acids, or at least 15 amino acids, or atleast 20 amino acids, or at least 30 amino acids, or at least 40 aminoacids, or at least 50 amino acids, or more, that are identical, orclosely similar, to target amino acids sequences.

In embodiments, the plurality of possible target flu viruses comprise atleast 5 possible target flu viruses, or at least 10 possible target fluviruses, or at least 15 possible target flu viruses, or at least 20possible target flu viruses, or at least 25 possible target flu viruses,or at least 30 possible target flu viruses, or at least 35 possibletarget flu viruses, or at least 40 possible target flu viruses, or atleast 45 possible target flu viruses, or at least 50 possible target fluviruses, or at least 55 possible target flu viruses, or at least 60possible target flu viruses, or at least 64 possible target flu viruses,or at least 65 possible target flu viruses, or more.

The assays and methods disclosed herein may be performed on a device, oron a system, for processing a sample. Such a sample may be asmall-volume clinical sample. The assays and methods disclosed hereincan be readily incorporated into and used in device for processing asample, or a system for processing a sample, which may be an automatedassay device, or may be an automated assay system. Such a device, andsuch a system, may be useful for the practice of the methods disclosedherein. For example, a device may be useful for receiving a sample. Adevice may be useful for preparing, or for processing a sample. A devicemay be useful for performing an assay on a sample. A device may beuseful for obtaining data from a sample. A device may be useful fortransmitting data obtained from a sample. A device may be useful fordisposing of a sample following processing or assaying of a sample.

The assays and methods disclosed herein can be readily incorporated intoand used in an automated assay device, and in an automated assay system.For example, systems as disclosed herein may include a communicationassembly for transmitting or receiving a protocol based on the analyteto be detected (e.g., a particular disease-causing agent or markerindicative of a particular disease-causing agent) or based on otheranalytes to be detected by the device or system. In embodiments, anassay protocol may be changed based on optimal scheduling of a pluralityof assays to be performed by a device, or may be changed based onresults previously obtained from a sample from a subject, or based onresults previously obtained from a different sample from the subject. Inembodiments, a communication assembly may comprise a channel forcommunicating information from said device to a computer, said whereinsaid channel is selected from a computer network, a telephone network, ametal communication link, an optical communication link, and a wirelesscommunication link. In embodiments, systems as disclosed herein maytransmit signals to a central location, or to an end user, and mayinclude a communication assembly for transmitting such signals. Systemsas disclosed herein may be configured for updating a protocol as neededor on a regular basis.

A device may be part of a system, a component of which may be a sampleprocessing device. A device may be a sample processing device. A sampleprocessing device may be configured to facilitate collection of asample, prepare a sample for a clinical test, or effect a chemicalreaction with one or more reagents or other chemical or physicalprocessing, as disclosed herein. A sample processing device may beconfigured to obtain data from a sample. A sample processing device maybe configured to transmit data obtained from a sample. A sampleprocessing device may be configured to analyze data from a sample. Asample processing device may be configured to communicate with anotherdevice, or a laboratory, or an individual affiliated with a laboratory,to analyze data obtained from a sample.

A sample processing device may be configured to be placed in or on asubject. A sample processing device may be configured to accept a samplefrom a subject, either directly or indirectly. A sample may be, forexample, a blood sample (e.g., a sample obtained from a fingerstick, orfrom venipuncture, or an arterial blood sample), a urine sample, abiopsy sample, a tissue slice, stool sample, or other clinical sample; awater sample, a soil sample, a food sample, an air sample; or othersample. A blood sample may comprise, e.g., whole blood, plasma, orserum. A sample processing device may receive a sample from the subjectthrough a housing of the device. The sample collection may occur at asample collection site, or elsewhere. The sample may be provided to thedevice at a sample collection site.

In some embodiments, a sample processing device may be configured toaccept or hold a cartridge. In some embodiments, a sample processingdevice may comprise a cartridge. The cartridge may be removable from thesample processing device. In some embodiments, a sample may be providedto the cartridge of the sample processing device. Alternatively, asample may be provided to another portion of a sample processing device.The cartridge and/or device may comprise a sample collection unit thatmay be configured to accept a sample.

A cartridge may include a sample, and may include reagents for use inprocessing or testing a sample, disposables for use in processing ortesting a sample, or other materials. Following placement of a cartridgeon, or insertion of a cartridge into, a sample processing device, one ormore components of the cartridge may be brought into fluid communicationwith other components of the sample processing device. For example, if asample is collected at a cartridge, the sample may be transferred toother portions of the sample processing device. Similarly, if one ormore reagents are provided on a cartridge, the reagents may betransferred to other portions of the sample processing device, or othercomponents of the sample processing device may be brought to thereagents. In some embodiments, the reagents or components of a cartridgemay remain on-board the cartridge. In some embodiments, no fluidics areincluded that require tubing or that require maintenance (e.g., manualor automated maintenance).

A sample or reagent may be transferred to a device, such as a sampleprocessing device. A sample or reagent may be transferred within adevice. Such transfer of sample or reagent may be accomplished withoutproviding a continuous fluid pathway from cartridge to device. Suchtransfer of sample or reagent may be accomplished without providing acontinuous fluid pathway within a device. In embodiments, such transferof sample or reagent may be accomplished by a sample handling system(e.g., a pipette); for example, a sample, reagent, or aliquot thereofmay be aspirated into an open-tipped transfer component, such as apipette tip, which may be operably connected to a sample handling systemwhich transfers the tip, with the sample, reagent, or aliquot thereofcontained within the tip, to a location on or within the sampleprocessing device. The sample, reagent, or aliquot thereof can bedeposited at a location on or within the sample processing device.Sample and reagent, or multiple reagents, may be mixed using a samplehandling system in a similar manner. One or more components of thecartridge may be transferred in an automated fashion to other portionsof the sample processing device, and vice versa.

A device, such as a sample processing device, may have a fluid handlingsystem. A fluid handling system may perform, or may aid in performing,transport, dilution, extraction, aliquotting, mixing, and other actionswith a fluid, such as a sample. In some embodiments, a fluid handlingsystem may be contained within a device housing. A fluid handling systemmay permit the collection, delivery, processing and/or transport of afluid, dissolution of dry reagents, mixing of liquid and/or dry reagentswith a liquid, as well as collection, delivery, processing and/ortransport of non-fluidic components, samples, or materials. The fluidmay be a sample, a reagent, diluent, wash, dye, or any other fluid thatmay be used by the device, and may include, but not limited to,homogenous fluids, different liquids, emulsions, suspensions, and otherfluids. A fluid handling system, including without limitation a pipette,may also be used to transport vessels (with or without fluid containedtherein) around the device. The fluid handling system may dispense oraspirate a fluid. The sample may include one or more particulate orsolid matter floating within a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipettetip, syringe, capillary, or other component. The fluid handling systemmay have portion with an interior surface and an exterior surface and anopen end. The fluid handling system may comprise a pipette, which mayinclude a pipette body and a pipette nozzle, and may comprise a pipettetip. A pipette tip may or may not be removable from a pipette nozzle. Inembodiments, a fluid handling system may use a pipette mated with apipette tip; a pipette tip may be disposable. A tip may form afluid-tight seal when mated with a pipette. A pipette tip may be usedonce, twice, or more times. In embodiments, a fluid handling system mayuse a pipette or similar device, with or without a pipette tip, toaspirate, dispense, mix, transport, or otherwise handle the fluid. Thefluid may be dispensed from the fluid handling system when desired. Thefluid may be contained within a pipette tip prior to being dispensed,e.g., from an orifice in the pipette tip. In embodiments, or instancesduring use, all of the fluid may be dispensed; in other embodiments, orinstances during use, a portion of the fluid within a tip may bedispensed. A pipette may selectively aspirate a fluid. The pipette mayaspirate a selected amount of fluid. The pipette may be capable ofactuating stirring mechanisms to mix the fluid within the tip or withina vessel. The pipette may incorporate tips or vessels creatingcontinuous flow loops for mixing, including of materials or reagentsthat are in non-liquid form. A pipette tip may also facilitate mixtureby metered delivery of multiple fluids simultaneously or in sequence,such as in 2-part substrate reactions.

The fluid handling system may include one or more fluidically isolatedor hydraulically independent units. For example, the fluid handlingsystem may include one, two, or more pipette tips. The pipette tips maybe configured to accept and confine a fluid. The tips may be fluidicallyisolated from or hydraulically independent of one another. The fluidcontained within each tip may be fluidically isolated or hydraulicallyindependent from one fluids in other tips and from other fluids withinthe device. The fluidically isolated or hydraulically independent unitsmay be movable relative to other portions of the device and/or oneanother. The fluidically isolated or hydraulically independent units maybe individually movable. A fluid handling system may comprise one ormore base or support. A base or support may support one or more pipetteor pipette units. A base or support may connect one or more pipettes ofthe fluid handling system to one another.

A sample processing device may be configured to perform processing stepsor actions on a sample obtained from a subject. Sample processing mayinclude sample preparation, including, e.g., sample dilution, divisionof a sample into aliquots, extraction, contact with a reagent,filtration, separation, centrifugation, or other preparatory orprocessing action or step. A sample processing device may be configuredto perform one or more sample preparation action or step on the sample.Optionally, a sample may be prepared for a chemical reaction and/orphysical processing step. A sample preparation action or step mayinclude one or more of the following: centrifugation, separation,filtration, dilution, enriching, purification, precipitation,incubation, pipetting, transport, chromatography, cell lysis, cytometry,pulverization, grinding, activation, ultrasonication, micro columnprocessing, processing with magnetic beads, processing withnanoparticles, or other sample preparation action or steps. For example,sample preparation may include one or more step to separate blood intoserum and/or particulate fractions, or to separate any other sample intovarious components. Sample preparation may include one or more step todilute and/or concentrate a sample, such as a blood sample, or otherclinical samples. Sample preparation may include adding ananti-coagulant or other ingredient to a sample. Sample preparation mayalso include purification of a sample. In embodiments, all sampleprocessing, preparation, or assay actions or steps are performed by asingle device. In embodiments, all sample processing, preparation, orassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample processing, preparation, or assayactions or steps are performed by a single device, and may be performedwithin a housing of a single device. In embodiments, many sampleprocessing, preparation, or assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

A sample processing device may be configured to run one or more assay ona sample, and to obtain data from the sample. An assay may include oneor more physical or chemical treatments, and may include running one ormore chemical or physical reactions. A sample processing device may beconfigured to perform one, two or more assays on a small sample ofbodily fluid. One or more chemical reaction may take place on a samplehaving a volume, as described elsewhere herein. For example one or morechemical reaction may take place in a pill having less than femtolitervolumes. In an instance, the sample collection unit is configured toreceive a volume of the bodily fluid sample equivalent to a single dropor less of blood or interstitial fluid. In embodiments, the volume of asample may be a small volume, where a small volume may be a volume thatis less than about 1000 μL, or less than about 500 μL, or less thanabout 250 μL, or less than about 150 μL, or less than about 100 μL, orless than about 75 μL, or less than about 50 μL, or less than about 40μL, or less than about 20 μL, or less than about 10 μL, or other smallvolume. In embodiments, all sample assay actions or steps are performedon a single sample. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all sample assayactions or steps are performed within a housing of a single device. Inembodiments, most sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, many sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

A sample processing device may be configured to perform a plurality ofassays on a sample. In embodiments, a sample processing device may beconfigured to perform a plurality of assays on a single sample. Inembodiments, a sample processing device may be configured to perform aplurality of assays on a single sample, where the sample is a smallsample. For example, a small sample may have a sample volume that is asmall volume of less than about 1000 μL, or less than about 500 μL, orless than about 250 μL, or less than about 150 μL, or less than about100 μL, or less than about 75 μL, or less than about 50 μL, or less thanabout 40 μL, or less than about 20 μL, or less than about 10 μL, orother small volume. A sample processing device may be capable ofperforming multiplexed assays on a single sample. A plurality of assaysmay be run simultaneously; may be run sequentially; or some assays maybe run simultaneously while others are run sequentially. One or morecontrol assays and/or calibrators (e.g., including a configuration witha control of a calibrator for the assay/tests) can also be incorporatedinto the device; control assays and assay on calibrators may beperformed simultaneously with assays performed on a sample, or may beperformed before or after assays performed on a sample, or anycombination thereof. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all of a plurality ofassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample assay actions or steps, of aplurality of assays, are performed by a single device, and may beperformed within a housing of a single device. In embodiments, manysample assay actions or steps, of a plurality of assays, are performedby a single device, and may be performed within a housing of a singledevice. In embodiments, sample processing, preparation, or assay actionsor steps may be performed by more than one device.

In embodiments, all of a plurality of assays may be performed in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about two hours, or less than aboutone hour, or less than about 40 minutes, or less than about 30 minutes,or less than about 25 minutes, or less than about 20 minutes, or lessthan about 15 minutes, or less than about 10 minutes, or less than about5 minutes, or less than about 4 minutes, or less than about 3 minutes,or less than about 2 minutes, or less than about 1 minute, or othershort time period.

A sample processing device may be configured to detect one or moresignals relating to the sample. A sample processing device may beconfigured to identify one or more properties of the sample. Forinstance, the sample processing device may be configured to detect thepresence or concentration of one analyte or a plurality of analytes or adisease condition in the sample (e.g., in or through a bodily fluid,secretion, tissue, or other sample). Alternatively, the sampleprocessing device may be configured to detect a signal or signals thatmay be analyzed to detect the presence or concentration of one or moreanalytes (which may be indicative of a disease condition) or a diseasecondition in the sample. The signals may be analyzed on board thedevice, or at another location. Running a clinical test may or may notinclude any analysis or comparison of data collected.

A chemical reaction or other processing step may be performed, with orwithout the sample. Examples of steps, tests, or assays that may beprepared or run by the device may include, but are not limited toimmunoassay, nucleic acid assay, receptor-based assay, cytometric assay,colorimetric assay, enzymatic assay, electrophoretic assay,electrochemical assay, spectroscopic assay, chromatographic assay,microscopic assay, topographic assay, calorimetric assay, turbidmetricassay, agglutination assay, radioisotope assay, viscometric assay,coagulation assay, clotting time assay, protein synthesis assay,histological assay, culture assay, osmolarity assay, and/or other typesof assays, centrifugation, separation, filtration, dilution, enriching,purification, precipitation, pulverization, incubation, pipetting,transport, cell lysis, or other sample preparation action or steps, orcombinations thereof. Steps, tests, or assays that may be prepared orrun by the device may include imaging, including microscopy, cytometry,and other techniques preparing or utilizing images. Steps, tests, orassays that may be prepared or run by the device may further include anassessment of histology, morphology, kinematics, dynamics, and/or stateof a sample, which may include such assessment for cells.

A device may be capable of performing all on-board steps (e.g., steps oractions performed by a single device) in a short amount of time. Adevice may be capable of performing all on-board steps on a singlesample in a short amount of time. For example, from sample collectionfrom a subject to transmitting data and/or to analysis may take about 3hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes orless, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute orless. The amount of time from accepting a sample within the device totransmitting data and/or to analysis from the device regarding such asample may depend on the type or number of steps, tests, or assaysperformed on the sample. The amount of time from accepting a samplewithin the device to transmitting data and/or to analysis from thedevice regarding such a sample may take about 3 hours or less, 2 hoursor less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes orless, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3minutes or less, 2 minutes or less, or 1 minute or less.

A device may be configured to prepare a sample for disposal, or todispose of a sample, such as a clinical sample, following processing orassaying of a sample.

In embodiments, a sample processing device may be configured to transmitdata obtained from a sample. In embodiments, a sample processing devicemay be configured to communicate over a network. A sample processingdevice may include a communication module that may interface with thenetwork. A sample processing device may be connected to the network viaa wired connection or wirelessly. The network may be a local areanetwork (LAN) or a wide area network (WAN) such as the Internet. In someembodiments, the network may be a personal area network. The network mayinclude the cloud. The sample processing device may be connected to thenetwork without requiring an intermediary device, or an intermediarydevice may be required to connect a sample processing device to anetwork. A sample processing device may communicate over a network withanother device, which may be any type of networked device, including butnot limited to a personal computer, server computer, or laptop computer;personal digital assistants (PDAs) such as a Windows CE device; phonessuch as cellular phones, smartphones (e.g., iPhone, Android, Blackberry,etc.), or location-aware portable phones (such as GPS); a roamingdevice, such as a network-connected roaming device; a wireless devicesuch as a wireless email device or other device capable of communicatingwireless with a computer network; or any other type of network devicethat may communicate possibly over a network and handle electronictransactions. Such communication may include providing data to a cloudcomputing infrastructure or any other type of data storageinfrastructure which may be accessed by other devices.

A sample processing device may provide data regarding a sample to, e.g.,a health care professional, a health care professional location, such asa laboratory, or an affiliate thereof. One or more of a laboratory,health care professional, or subject may have a network device able toreceive or access data provided by the sample processing device. Asample processing device may be configured to provide data regarding asample to a database. A sample processing device may be configured toprovide data regarding a sample to an electronic medical records system,to a laboratory information system, to a laboratory automation system,or other system or software. A sample processing device may provide datain the form of a report.

A laboratory, device, or other entity or software may perform analysison data regarding a sample in real-time. A software system may performchemical analysis and/or pathological analysis, or these could bedistributed amongst combinations of lab, clinical, and specialty orexpert personnel. Analysis may include qualitative and/or quantitativeevaluation of a sample. Data analysis may include a subsequentqualitative and/or quantitative evaluation of a sample. Optionally, areport may be generated based on raw data, pre-processed data, oranalyzed data. Such a report may be prepared so as to maintainconfidentiality of the data obtained from the sample, the identity andother information regarding the subject from whom a sample was obtained,analysis of the data, and other confidential information. The reportand/or the data may be transmitted to a health care professional. Dataobtained by a sample processing device, or analysis of such data, orreports, may be provided to a database, an electronic medical recordssystem, to a laboratory information system, to a laboratory automationsystem, or other system or software.

Nucleic Acid Detection

Markers indicative of a disease, such as a respiratory disease, includenucleic acid markers. Such nucleic acid markers include, for example,viral nucleic acids, or portions thereof; bacterial nucleic acids, orportions thereof; and nucleic acids, or portions thereof, derived fromother micro-organisms. Methods for detecting nucleic acid markers in asample, including in a small volume sample, include methods in whichsmall amounts of nucleic acid may be amplified (e.g., copies made). Forexample, polymerase chain reaction (PCR) and related methods are commonmethods of nucleic acid amplification. PCR is discussed, for example, inU.S. Pat. No. 4,683,195; and generally in Mullis et al., Cold SpringHarbor Symposium on Quantitative Biology, volume 51:263 (1987); Erlich,ed., PCR Technology (Stockton Press, N Y, 1989). PCR is one, but not theonly, example of a nucleic acid polymerase reaction method foramplifying a nucleic acid test sample comprising the use of a knownnucleic acid as a primer and a nucleic acid polymerase to amplify orgenerate a specific piece of nucleic acid. Further discussion of PCR andother methods may be found, for example, in Molecular Cloning ALaboratory Manual by Green and Sambrook, Cold Spring Harbor LaboratoryPress, 4^(th) Edition 2012, which is incorporated by reference herein inits entirety. PCR and many other amplification methods must be performedat multiple different temperatures, requiring repeated temperaturechanges during the PCR reaction (“thermal cycling”). Other amplificationmethods, such as, e.g., loop-mediated isothermal amplification (“LAMP”)(see, e.g. U.S. Pat. No. 6,410,278), and other methods, includingmethods discussed below, require less or less extensive thermal cyclingthan does PCR, or require no thermal cycling.

Nucleic Acid Amplification and Detection Methods without Thermal Cycling

Methods for nucleic acid amplification which do not require thermalcycling are described in U.S. Patent Application 61/800,606, filed Mar.15, 2013, incorporated by reference herein in its entirety. Such methodsmay be used to detect nucleic acid markers of disease, such asrespiratory disease, in small-volume samples in short periods of time.Such methods are discussed below, and examples of results obtained withsuch methods, from small samples and in short periods of time, arepresented in the Figures and Examples disclosed herein. In thefollowing, such methods are termed “non-cycling amplification methods.”

Non-cycling amplification methods of nucleic acid amplification may beapplied to double-stranded DNA. However, target nucleic acid moleculesneed not be limited to double-stranded DNA targets; for example,double-stranded DNA for use in non-cycling amplification methodsdescribed herein may be prepared from viral RNA, or mRNA, or othersingle stranded RNA target sources, by reverse transcriptase. In furtherexample, double-stranded DNA for use in non-cycling amplificationmethods described herein may be prepared from single-stranded DNAtargets by DNA polymerase. Such methods may be applied as an initialstep, prior to application of the non-cycling amplification methodsdiscussed below.

Amplification of a double-stranded DNA target, for example, begins witha primary double-stranded DNA to be amplified (termed the “primarynucleic acid” in the following). The primary nucleic acid contains atarget region termed a template region; the template region has atemplate sequence. Such a double-stranded template region contains afirst DNA strand and a complementary second DNA strand, and includes a5′ terminal nucleotide in one strand and a 3′ terminal nucleotide in theother strand that are complementary to each other.

A first primer and a second primer are provided which each havetemplate-binding regions and tail regions; the primer template-bindingregions are complementary to the target template regions. The tailregions of the primers may contain three components: a) the 5′ terminalnucleotide of the primer, b) an innermost nucleotide, wherein theinnermost nucleotide is downstream from the 5′ terminal nucleotide, andc) a middle section between the 5′ terminal nucleotide and the innermostnucleotide, comprising one or more nucleotides. In addition, at leastportions of the two primer tail regions may be complementary to eachother when properly aligned.

It should be noted that although the tail region of the second primermay contain a nucleotide sequence which is complementary to thenucleotide sequence of the tail region of the first primer, typically,products formed by the annealing of the first primer and second primerare not desirable or useful for methods or compositions provided herein.Accordingly, in some embodiments, steps may be taken to minimize theformation of first primer-second primer annealed products. Such stepsmay include, for example, not pre-incubating a first primer and a secondprimer under conditions where the primers may anneal for an extendedperiod of time before initiating a method provided herein.

The primary nucleic acid may be treated with a polymerase and a firstcopy of the first primer under conditions such that the template-bindingregion of the first copy of the first primer anneals to the first strandof the nucleic acid template. Under these conditions, an extensionproduct of the first copy of the first primer is formed. The polymerase,which may have strand displacement activity, may catalyze the formationof the extension product of the first copy of the first primer. Thefirst copy of the first primer may be covalently linked to thesynthesized extension product, such that the first copy of the firstprimer (which is complementary to the first strand of the nucleic acidtemplate) becomes part of the molecule described herein as the“extension product of the first copy of the first primer.” Thetemplate-binding region but not the tail region of the first copy of thefirst primer anneals to the first strand of the nucleic acid template.Examples of conditions suitable for polymerase-based nucleic acidsynthesis are known in the art and are provided, for example, inMolecular Cloning: A Laboratory Manual, M. R. Green and J. Sambrook,Cold Spring Harbor Laboratory Press (2012), which is incorporated byreference herein in its entirety.

The extension product of the first copy of the first primer may betreated with a polymerase (which may have strand displacement activity)and with the second primer under conditions such that thetemplate-binding region of the second primer anneals to the extensionproduct of the first copy of the first primer. In this way, an extensionproduct of the second primer may be formed. The polymerase may displacethe first strand of the nucleic acid template from the extension productof the first copy of the first primer during the synthesis of theextension product of the second primer. The second primer may becovalently linked to the synthesized extension product, such that thesecond primer becomes part of the molecule described herein as the“extension product of the second primer.” The extension product of thesecond primer is complementary to the extension product of the firstcopy of the first primer. The template-binding region but not the tailregion of the second primer may anneal to the extension product of thefirst copy of the first primer when the second primer anneals to theextension product of the first copy of the first primer.

The extension product of the second primer may be treated with apolymerase (which may have strand displacement activity) and a secondcopy of the first primer so as to form an extension product of thesecond copy of the first primer. During the generation of the extensionproduct of the second copy of the first primer, the second copy of thefirst primer may be covalently linked to the synthesized extensionproduct, such that the second copy of the first primer becomes part ofthe molecule described herein as the “extension product of the secondcopy of the first primer.” The extension product of the second copy ofthe first primer is complementary to the extension product of the secondprimer.

Generation of the extension product of the second copy of the firstprimer may result in the generation of a molecule comprising theextension product of the second copy of the first primer and theextension product of the second primer, which may be referred to hereinas the “secondary nucleic acid.” A secondary nucleic acid may comprisethe 3′ terminal region of the extension product of the second primer(and the complement thereof) and may comprise the 3′ terminal region ofthe extension product of the second copy of the first primer (and thecomplement thereof). Secondary nucleic acid molecules include sequencesof the template region adjacent to tail sequences. In embodiments,double-stranded nucleic acids are produced in which complementarytemplate and tail region sequences line up. In practice, multiple copies(e.g., two or more) of the secondary nucleic acid are produced by anyprocess whereby a nucleic acid having the general structure of thesecondary nucleic acid may be generated, including by practice ofnon-cycling amplification methods discussed herein.

Thus, pairs of copies of the secondary nucleic acid may be provided.Further numbers of copies may then be generated, for example, byrepetition of the foregoing steps and methods. For example, the fullprocess as described above for generating a secondary nucleic acid froma primary nucleic acid may be repeated two times, in order to generate atwo pairs of copies of the secondary nucleic acid; further repetitionsmay be performed to amplify the number of copies further, e.g., toexponentially amplify the number of copies (e.g., by powers of two).

In addition, since the secondary nucleic acid molecules includesequences of the template region adjacent to tail sequences, partiallydouble-stranded nucleic acids may be produced in which tail regionsequences hybridize and line up. Since these tail region sequences areattached to single-stranded template regions, a cross-over structurehaving two nucleic acid strands together held by the hybridized tailregion sequences is produced. These cross-over structures may beextended by a polymerase to form extension products of both componentstrands. These extension products which may be referred to as“concatemer strands.” Two concatemer strands may be annealed together,and may be collectively referred to as a concatemer; such concatemersmay contain two or more copies of the nucleic acid template.

In some embodiments, even longer concatemers may be formed. For example,concatemers may anneal together; or two concatemer molecules may form across-over structure similar to those formed by the shorter moleculestermed concatemer strands, as discussed above, followed by a largerconcatemer molecule containing four copies of the nucleic acid template.In another example, a secondary nucleic acid and a concatemer may form across-over structure, followed by a larger concatemer moleculecontaining three copies of the nucleic acid template. In someembodiments, multiple different concatemers of multiple differentlengths may be simultaneously generated.

Thus, concatemers generated according to such methods may be of anylength of nucleotides. In some embodiments, concatemer moleculesgenerated herein may be at least 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000nucleotides in length. Concatemers generated according to such methodsmay contain any number of copies of a nucleic acid template. In someembodiments, concatemer molecules generated herein may contain at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies of a nucleic acidtemplate. Further examples are provided, and greater detail of these andother examples, is provided in U.S. Patent Application 61/800,606, filedMar. 15, 2013.

Detection of Reactions

Progress of a method provided herein may be monitored in multipledifferent ways. In one embodiment, a reaction may be assayed for anucleic acid amplification product (e.g. for the level of the product orthe rate of its generation). In another embodiment, a reaction may beassayed for the activity of a polymerase along a nucleic acid template(e.g. for movement of a polymerase along a template strand). Thus, insome embodiments, events of a method provided herein may observed due tothe accumulation of product from a method (which may be during or aftercompletion of steps of the method), or due to detectable eventsoccurring during the steps of a method.

The presence of amplified nucleic acids can be assayed, for example, bydetection of reaction products (amplified nucleic acids or reactionby-products) or by detection of probes associated with the reactionprogress.

In some embodiments, reaction products may be identified by staining theproducts with a dye. In some embodiments, a dye may have greaterfluorescence when bound to a nucleic acid than when not bound to anucleic acid. In some embodiments, a dye may intercalate with adouble-stranded nucleic acid or it may bind to an external region of anucleic acid. Nucleic acid dyes that may be used with methods andcompositions provided herein include, for example, cyanine dyes,PicoGreen®, OliGreen®, RiboGreen®, SYBR® dyes, SYBR® Gold, SYBR® GreenI, SYBR® Green II, ethidium bromide, dihydroethidium, BlueView™, TOTO®dyes, TO-PRO® dyes, POPO® dyes, YOYO® dyes, BOBO® dyes, JOJO® dyes,LOLO® dyes, SYTOX® dyes, SYTO® dyes, propidium iodide, hexidium iodide,methylene blue, DAPI, acridine orange, quinacrine, acridine dimers,9-amino-6-chloro-2-methoxyacridine, bisbenzimide dyes, Hoechst dyes,7-aminoactinomycin D, actinomycin D, hydroxystilbamidine, pyronin Y,Diamond™ dye, GelRed™, GelGreen™ and LDS 751.

In some embodiments, reaction products may be identified by analysis ofturbidity of amplification reactions for example, where increasedturbidity is correlated with formation of reaction products and reactionby-products (e.g. pyrophosphate complexed with magnesium).

In some embodiments, reaction products may be identified by separating areaction performed according to a method herein by gel electrophoresis,followed by staining of the gel with a dye for nucleic acids. The dyemay be any nucleic acid dye disclosed herein or otherwise known in theart.

In some embodiments, any method or composition known in the art for thedetection of nucleic acids or processes associated with the generationof nucleic acids may be used with methods and compositions providedherein.

In some embodiments, a nucleic acid probe which contains a nucleotidesequence complementary to a portion of a nucleic acid template strand(or strand having a similar or identical sequence) and which containsone or both of a fluorescent reporter (fluorophore) and a quencher areincluded in a reaction provided herein.

In an example, a nucleic acid probe may contain a fluorescent reporterat its 5′ or 3′ terminus, and a quencher at the other terminus.

In another example, a nucleic acid probe may contain a fluorescentreporter at its 5′ or 3′ terminus, and it may be annealed to a nucleicacid primer containing a quencher. The nucleic acid primer containing aquencher may contain the quencher at a position in the primer such thatwhen the nucleic acid probe is annealed to the primer, the fluorescentreporter is quenched.

In probes containing a fluorescent reporter and quencher pair, thefluorescent reporter and quencher may be selected so that the quenchercan effectively quench the reporter. In some embodiments, a fluorescentreporter is paired with a quencher where the emission maximum of thefluorescent reporter is similar to the absorption maximum of thequencher. Fluorphores that may be used as the fluorescent reporterinclude, for example, CAL Fluor Gold, CAL Fluor Orange, Quasar 570, CALFluor Red 590, CAL Fluor Red 610, CAL Fluor Red 610, CAL Fluor Red 635,Quasar 670 (Biosearch Technologies), VIC, NED (Life Technologies), Cy3,Cy5, Cy5.5 (GE Healthcare Life Sciences), Oyster 556, Oyster 645(Integrated DNA Technologies), LC red 610, LC red 610, LC red 640, LCred 670, LC red 705 (Roche Applies Science), Texas red, FAM, TET, HEX,JOE, TMR, and ROX. Quenchers that may be used include, for example,DDQ-I, DDQ-II (Eurogentec), Eclipse (Epoch Biosciences), Iowa Black FQ,Iowa Black RQ (Integrated DNA Technologies), BHQ-1, BHQ-2, BHQ-3(Biosearch Technologies), QSY-7, QSY-21 (Molecular Probes), and Dabcyl.

In some embodiments, a method provided herein may be monitored in anapparatus containing a light source and an optical sensor. In somesituations, the reaction may be positioned in the path of light from thelight source, and light absorbed by the sample (e.g. in the case of aturbid reaction), scattered by the sample (e.g. in the case of a turbidreaction), or emitted by the sample (e.g. in the case of a reactioncontaining a fluorescent molecule) may be measured. In some embodiments,a method provided herein may be performed or monitored in a device ormodule therein as disclosed in U.S. patent application Ser. No.13/769,779, filed Feb. 18, 2013, which is herein incorporated byreference in its entirety.

Detection of Nucleic Acids and Protein Markers of Infection

A sample, such as a throat swab, a nasal swab, a mouth swab (e.g., acheek swab), a vaginal swab, saliva, blood, or other sample, may be usedfor more than one assay. For example, a sample may be subjected tonucleic acid testing and to testing for peptides indicative of aninfection. In embodiments, a sample may be divided into two or moreportions, and each portion may be subjected to a single test, or may besubjected to a plurality of tests. Nucleic acid testing may be used toidentify nucleic acid molecules, or portions thereof, whose presenceindicates the presence of disease-causing organisms (e.g., viruses,bacteria, and other organisms which carry such nucleic acids). Protein(or peptide) testing may be used to identify peptides or proteins, orportions thereof, whose presence indicates the presence ofdisease-causing organisms (e.g., organisms which express such proteins,or peptides). Protein or peptide testing may be used to identifydisease-causing organisms (e.g., viruses, bacteria, and other organisms)in a sample, and may also be used to identify antibodies directedagainst such agents that may be present in a sample. Thus, forms ofprotein (or peptide) testing include testing for the presence ofantibodies to targets whose presence indicates the presence ofdisease-causing organisms. Such nucleic acid and protein (or peptide)testing may be used to identify, or estimate, or otherwise determine atwhat stage an infection in a subject is at the time the sample wastaken, by detecting, or determining the amounts of, or both, bothnucleic acid markers indicative of a particular infection and protein(or peptide) markers indicative of the same infection (such proteinmarkers of the same infection include antibodies to the micro-organismthat causes the infection, as well as protein markers present on or inthe micro-organism itself).

Nucleic acid markers of an infection include DNA and RNA molecules, andfragments thereof, unique to the infectious agent (e.g., viral nucleicacids, or bacterial nucleic acids, or other nucleic acids of any otherinfectious micro-organism). Peptide or protein markers of an infectioninclude peptides or proteins unique to the infectious agent (e.g.,bacterial peptides); cytokines and other peptides produced in responseto the infection; and antibodies produced in response to the infection.

For example, where nucleic acid markers indicative of a particularinfection are relatively numerous, while antibody markers indicative ofthat particular infection are relatively sparse, then the infection is arecent infection; however, where nucleic acid markers indicative of aparticular infection are relatively numerous, and antibody markersindicative of that particular infection are also relatively numerous,then the infection is not a recent infection, since the subject has hadthe time to produce infection-specific antibodies. Where nucleic acidmarkers indicative of a particular infection are relatively sparse, andantibody markers indicative of that particular infection are relativelynumerous, then the infection may be waning and in a late stage. Otherprotein markers (other than antibodies to the infectious organism),being produced by the disease-causing organism itself, such as viralcoat proteins, bacterial cell wall proteins, bacterial toxins, and othernon-antibody markers, typically follow a time-course more similar tothat of nucleic acid markers of a particular infection and less similarto that of antibody markers of a particular infection.

A typical response in human subjects to infection by many infectiousagents includes increased levels of inflammatory cytokines (includinginterleukin 1 (IL-1), IL-6, IL-18, tumor necrosis factor α (TNF-α),gamma interferon (IFN-γ), and others). Cytokine levels may increaserapidly upon infection.

The time-course of production of antibodies to an infectious agent(e.g., a virus, bacteria, or other infectious micro-organism) variesbetween individual subjects and from infection to infection; suchtime-courses may be known or identified for different kinds ofinfections. In general, a few days or more are required beforeantibodies to an infectious agent are detectable in a subject; oncedetectable, the amount of antibodies detected in a subject grows, oftenvery rapidly, and may plateau (or peak) over a period of weeks or monthsfollowing infection. In addition to the type of infection, factors whichmay affect the plateau (or peak) levels, and the timing at which theselevels are reached, include whether or not the infection is acute orchronic; the severity of the infection; other diseases or conditionsaffecting the subject; the nutritional status of the subject;environmental factors; and other factors.

The time-course of an acute infection may be short; for example, anacute infection may follow a time-course measured in days or weeks. Forexample, many viral and bacterial infections in an otherwise healthyhuman subject typically resolve within about a week. Levels of nucleicacid and protein markers indicative of viral, bacterial, and otherinfections typically rise, and then fall, during the course of theinfection. Initially, upon infection, and closely following the time ofinfection, markers of the infectious agent (e.g., nucleic acid markersand protein markers indicative of the viral, bacterial, or otherinfection) will be detectable in samples obtained from a subject; thelevels of such markers will rise from the time of infection, and willtypically peak within a few days (for a short-lived infection) or withina few weeks (for an infection of longer duration). Antibodies to theinfectious agent may be detected within a week or two following theinfection, and may then further increase for several weeks (e.g., for amonth or more). If the infection itself resolves and the infectiousagent is cleared from the subject, the levels of antibodies will thenslowly decline over a period of months.

Longer term, or chronic infections may follow a longer time-course. Forexample, the time-course of viral markers and antibody formation in aperson infected with the human immunodeficiency virus (HIV) may follow atime-course over many months and years. Initially, HIV viral markers(e.g., the viral p24 antigen, viral nucleic acids, and other viralmarkers produced by the virus itself) may be present (in samplesobtained from a subject) in high levels for the first few monthsfollowing infection, and may peak by about 6 months after infection. Incontrast, anti HIV antibodies (e.g., antibodies to gp120 or other viralantigenic epitopes) are not detectable in samples obtained from asubject for a few months following infection, but become detectable byabout 3-5 months after infection, and anti-HIV antibody levels riserapidly over the following 6 months or so, continuing to rise at a lessrapid rate from about 1 year after infection to about 4 to 6 yearsfollowing infection. Over this period of time (from about 1 year toabout 5 years) the viral marker levels may be very low; however, in theabsence of treatment, as T-cell levels (e.g., CD4 T-cells) willtypically have been falling over the period of time from about 1 year toabout 5 years following HIV infection, the subject may begin to sufferfrom systemic immune deficiency and further T-cell loss (e.g., CD-8T-cells) about 5-6 years following HIV infection. Levels of HIV viralmarkers will typically rise as systemic immune deficiency becomesapparent 5-6 years following HIV infection.

The time-courses relevant to the detection of marker may also depend onthe type of sample tested for the presence of the marker. For example,in some infections, the causative organism, and nucleic acid and proteinmarkers of the organism, may be found initially in blood, or saliva, orother fluid or tissue; and may later on be found in urine or stoolsamples; and even later on may be detectable in tissue samples.Antibodies to such disease-causing organisms, which typically appearsome time following the appearance of the organism itself in samples,may found first in blood, and then, following their appearance in blood,in saliva, urine, or stool.

Devices

The devices, systems, and assays disclosed herein may utilizetechniques, devices, systems, and assays disclosed, for example, in U.S.Pat. No. 8,088,593; U.S. Pat. No. 8,380,541; U.S. patent applicationSer. No. 13/769,798, filed Feb. 18, 2013; U.S. patent application Ser.No. 13/769,779, filed Feb. 18, 2013; U.S. Application Ser. No.61/800,606, filed Mar. 15, 2013; U.S. Application Ser. No. 61/766,095,filed Feb. 18, 2013; and U.S. Patent Application 61/805,923, filed Mar.27, 2013, all of which are incorporated by reference herein (supra).

For example, devices for use in performing assays for detecting aplurality of disease-causing agents in a single clinical sample, or in aplurality of aliquots of a single clinical sample include cartridgesincluding some or all of reagent vessels, reaction vessels, tools andimplements used in assays, and reagents used in assays. A cartridge maycontain one or more of: reagent vessels; reaction vessels; cytometrycuvettes; waste containers; sample collection devices or samplecollection vessels; and other vessels and materials. Such devices mayinclude multiple vessels containing reagents for use in an assay for thedetection of a plurality of markers indicative of an infectious agent,e.g., an upper respiratory infectious agent; a lower respiratoryinfectious agent; a sexually transmitted disease-causing agent; an agentdetectable from a sample obtained from a swab (e.g., a throat swab, anasal swab, a mouth swab (e.g., a cheek swab), a vaginal swab, or otherswab); an agent detectable from a blood sample; or combinations thereof.

Thus, for example, a cartridge may contain a plurality of reagentvessels; a plurality of reagent vessels containing reagents fordetecting a marker indicative of a disease-causing agent.Disease-causing agents may include agents which cause upper respiratorydisorders, or lower respiratory disorders, or sexually transmitteddiseases. A disease-causing agent may be detected in a blood sample. Adisease-causing agent may be detected in a sample obtained with a swab,such as a throat swab, or a nasal swab, or a mouth swab (e.g., a cheekswab), or a vaginal swab, or other sample, or combinations thereof.

In embodiments, a device may be or comprise a cartridge configured tocontain one or more reagent vessels, and one or more reaction vessels,e.g., for use in nucleic acid assays; for use in immunoassays (e.g.,ELISA assays); for use in general chemistry assays (e.g., for clinicalelectrolytes, vitamin levels, blood component levels, and othertargets); for use in cytometric assays; and for combinations thereof. Inembodiments, such a device may include reagents, reaction vessels, andtools, cuvettes, and other implements for use in nucleic acid assays;for immunoassays (e.g., ELISA assays); general chemistry assays (e.g.,for clinical electrolytes, vitamin levels, blood component levels, andother targets); cytometric assays; and for combinations thereof.

In embodiments, a cartridge for use in performing assays for detecting aplurality of disease-causing agents as disclosed herein may include oneor more spaces or vessels for holding a swab or swabs. A single swab maybe placed in a single space, or in a single vessel; in embodiments, twoswabs may be placed in a single space, or single vessel. In embodiments,a plurality of swabs may be placed in a single space, or single vessel.Vessels for holding a swab, or swabs, may contain a reagent, or adiluent, or other solution for use with a swab or swabs.

A nasal swab may be useful for testing for upper respiratory diseases,and a throat swab may be useful for testing lower respiratory diseases.In embodiments, a mouth swab (e.g., a cheek swab, a tongue swab, a gumswab, or other swab taken within the mouth) may be used in addition to,or in place of, a nasal or throat swab. Nasal and throat swabs may beobtained from a single patient, and may be analyzed at the same time, orat nearly the same time. For example, a throat swab may be placed in onevessel in a cartridge, and a nasal swab may be placed in a differentvessel in the cartridge, for analysis in an analysis device or analysissystem. For example, a mouth swab may be placed in one vessel in acartridge, and a nasal swab may be placed in a different vessel in thecartridge, for analysis in an analysis device or analysis system. Forexample, a throat swab may be placed in one vessel in a cartridge, and amouth swab may be placed in a different vessel in the cartridge, foranalysis in an analysis device or analysis system. These vessels maycontain a reagent, or a diluent, or other solution for use with theswabs; such reagents may be different for the throat swab and the nasalswab. In embodiments, reagents may be different for a mouth swab thanfor a throat swab or nasal swab. In a further example, a throat swab anda nasal swab from a single subject may be placed in the same vessel in adevice. In a further example, a mouth swab and a nasal swab from asingle subject may be placed in the same vessel in a device. In afurther example, a throat swab and a mouth swab from a single subjectmay be placed in the same vessel in a device. The vessel may contain areagent, or a diluent, or other solution for use with these swabs. Thedevice may be placed in an analysis device, or within an analysissystem, for analysis. Such analysis devices and analysis systems may beplaced at the same location as that where the sample was obtained; orsuch analysis devices and analysis systems may be at a differentlocation or locations than the location where the sample was obtained.

In embodiments, a cartridge for use in performing assays for detecting aplurality of disease-causing agents as disclosed herein may include oneor more spaces or vessels for holding a blood sample. In embodiments, acartridge for use in performing assays for detecting a plurality ofdisease-causing agents as disclosed herein may include one or morespaces or vessels for holding a blood sample and may also include one ormore spaces or vessels for holding a swab or swabs. Thus, a device, suchas a cartridge, may hold a blood sample and a throat swab; may hold ablood sample and a nasal swab; and may hold a blood sample, a throatswab, and a nasal swab. In embodiments, a device, such as a cartridge,may hold a blood sample and a mouth swab; may hold a blood sample, amouth swab, and a nasal swab; and may hold a blood sample, a mouth swab,a throat swab, and a nasal swab. Similarly, a device, such as acartridge, may hold a blood sample and a vaginal swab; and may hold ablood sample, and one or more of a mouth swab, a throat swab, a nasalswab, and a vaginal swab.

In embodiments, a cartridge for use in performing assays for detecting aplurality of disease-causing agents as disclosed herein may include oneor more spaces or vessels for holding a urine sample. In embodiments, acartridge for use in performing assays for detecting a plurality ofdisease-causing agents as disclosed herein may include one or morespaces or vessels for holding a urine sample and may also include one ormore spaces or vessels for holding a swab or swabs. Thus, a device, suchas a cartridge, may hold a urine sample and a throat swab; may hold aurine sample and a nasal swab; may hold a urine sample, a throat swab,and a nasal swab; may hold a urine sample, a throat swab, a mouth swab,and a nasal swab; and may hold a urine sample, and one or more of athroat swab, a mouth swab, a vaginal swab, and a nasal swab.

It will be understood that such devices, such as cartridges, may holdother types of samples as well, and that any combination of types ofsamples may be held by such devices (e.g., cartridges). In any and allsuch cases, the sample or samples may be analyzed in a sample analysisdevice or a sample analysis system.

A cartridge may include a sample, and may include reagents for use inprocessing or testing a sample, disposables for use in processing ortesting a sample, or other materials. Following placement of a cartridgeon, or insertion of a cartridge into, a sample processing device orsystem, one or more components of the cartridge may be brought intofluid communication with other components of the sample processingdevice. For example, if a sample is collected at a cartridge, the samplemay be transferred to other portions of the sample processing device.Similarly, if one or more reagents are provided on a cartridge, thereagents may be transferred to other portions of the sample processingdevice, or other components of the sample processing device may bebrought to the reagents. In some embodiments, the reagents or componentsof a cartridge may remain on-board the cartridge. In some embodiments,no fluidics are included that require tubing or that require maintenance(e.g., manual or automated maintenance).

A sample or reagent may be transferred to a device, such as a sampleprocessing device. A sample or reagent may be transferred within adevice. Such transfer of sample or reagent may be accomplished withoutproviding a continuous fluid pathway from cartridge to device. Suchtransfer of sample or reagent may be accomplished without providing acontinuous fluid pathway within a device. In embodiments, such transferof sample or reagent may be accomplished by a sample handling system(e.g., a pipette); for example, a sample, reagent, or aliquot thereofmay be aspirated into an open-tipped transfer component, such as apipette tip, which may be operably connected to a sample handling systemwhich transfers the tip, with the sample, reagent, or aliquot thereofcontained within the tip, to a location on or within the sampleprocessing device. The sample, reagent, or aliquot thereof can bedeposited at a location on or within the sample processing device.Sample and reagent, or multiple reagents, may be mixed using a samplehandling system in a similar manner. One or more components of thecartridge may be transferred in an automated fashion to other portionsof the sample processing device, and vice versa.

As shown in FIG. 20A, FIG. 20B, and FIG. 20C, a vessel for holding aswab may be loaded onto a cartridge, where it may be retained untilneeded for analysis; the cartridge may be loaded onto an analysis deviceor analysis system, thereby loading the swab (and any other samples orsample containers on the cartridge as well). As shown in FIG. 20A, avessel for holding a swab (a swab vessel 10) may be loaded onto acartridge 20 by placement into a receptacle 30. The cartridge 20 asshown also includes cavities and wells 40 for receiving and holdingreagents and vessels. A swab vessel 10 may contain a swab in placewithin the swab vessel 10, or may be loaded onto a cartridge without aswab in place within the swab vessel 10.

As shown in FIG. 20B, a vessel for holding a swab (a swab vessel 10) maybe loaded onto a cartridge 20 by placement into a receptacle 30. Thecartridge 20 as shown also includes cavities and wells 40 for receivingand holding reagents and vessels. In the embodiment shown in FIG. 20B,the cartridge 20 also includes a sample collection vessel 50, which mayhold, e.g., blood, urine, or other sample. The arrows leading away fromthe swab vessel 10 indicate how the swab vessel 10 may be placed into areceptacle 30 in the cartridge 20.

As shown in FIG. 20C, a vessel for holding a swab (a swab vessel 10) maybe loaded onto a cartridge 20 by placement into a receptacle 30. Thecartridge 20 as shown also includes cavities and wells 40 for receivingand holding reagents and vessels. As shown in the embodiment of FIG.20C, the cartridge 20 includes a swab receptacle 60 configured to hold aswab 70. In embodiments (e.g., in the embodiment illustrated in FIG.20C) a cartridge 20 having a swab receptacle 60 may optionally include asample collection vessel 50, which may hold, e.g., blood, urine, orother sample. Such a swab 70 may be held in swab receptacle 60 prior toits use in collecting a sample. In embodiments, a swab 70 may be placedwithin a swab vessel 10 after collection of a sample with swab 70. Inthe embodiment shown in FIG. 20C, swab vessel 10 may be loaded onto acartridge without a swab in place within the swab vessel 10 prior to useof swab 70, and swab vessel 10 may be replaced in a receptacle 30,holding swab 70 within swab vessel 10 after collection of a sample byswab 70.

Swabs may be any suitable swab for collection of a sample. Severalexamples of swabs suitable for use in sample collection are shown inFIG. 21. Swabs with flocked tips (swab 100, and the shorter swab 200,suitable for pediatric use), or those also suitable for use inestablishing cultures of material obtained by swabbing a body orifice, abody cavity or surface of a subject (swab 300), cotton-tipped swabs(swab 400), and other swabs may be used to collect a sample from apatient for use with the methods, systems, and devices disclosed herein.For example, samples may be obtained by swabbing a nasal cavity, athroat, a mouth, a vagina, or other orifice, body cavity, or location onor within a subject.

Systems

An analysis system, which may include an analysis device, such as asample processing device, may have a fluid handling system (also termedherein a sample handling system). A fluid handling system may perform,or may aid in performing, transport, dilution, extraction, aliquotting,mixing, and other actions with a fluid, such as a sample. In someembodiments, a fluid handling system may be contained within a devicehousing. A fluid handling system may permit the collection, delivery,processing and/or transport of a fluid, dissolution of dry reagents,mixing of liquid and/or dry reagents with a liquid, as well ascollection, delivery, processing and/or transport of non-fluidiccomponents, samples, or materials. The fluid may be a sample, a reagent,diluent, wash, dye, or any other fluid that may be used by the device,and may include, but not limited to, homogenous fluids, differentliquids, emulsions, suspensions, and other fluids. A fluid handlingsystem, including without limitation a pipette, may also be used totransport vessels (with or without fluid contained therein) around thedevice. The fluid handling system may dispense or aspirate a fluid. Thesample may include one or more particulate or solid matter floatingwithin a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipettetip, syringe, capillary, or other component. The fluid handling systemmay have portion with an interior surface and an exterior surface and anopen end. The fluid handling system may comprise a pipette, which mayinclude a pipette body and a pipette nozzle, and may comprise a pipettetip. A pipette tip may or may not be removable from a pipette nozzle. Inembodiments, a fluid handling system may use a pipette mated with apipette tip; a pipette tip may be disposable. A tip may form afluid-tight seal when mated with a pipette. A pipette tip may be usedonce, twice, or more times. In embodiments, a fluid handling system mayuse a pipette or similar device, with or without a pipette tip, toaspirate, dispense, mix, transport, or otherwise handle the fluid. Thefluid may be dispensed from the fluid handling system when desired. Thefluid may be contained within a pipette tip prior to being dispensed,e.g., from an orifice in the pipette tip. In embodiments, or instancesduring use, all of the fluid may be dispensed; in other embodiments, orinstances during use, a portion of the fluid within a tip may bedispensed. A pipette may selectively aspirate a fluid. The pipette mayaspirate a selected amount of fluid. The pipette may be capable ofactuating stirring mechanisms to mix the fluid within the tip or withina vessel. The pipette may incorporate tips or vessels creatingcontinuous flow loops for mixing, including of materials or reagentsthat are in non-liquid form. A pipette tip may also facilitate mixtureby metered delivery of multiple fluids simultaneously or in sequence,such as in 2-part substrate reactions.

A fluid handling system may include one or more fluidically isolated orhydraulically independent units. For example, the fluid handling systemmay include one, two, or more pipette tips. The pipette tips may beconfigured to accept and confine a fluid. The tips may be fluidicallyisolated from or hydraulically independent of one another. The fluidcontained within each tip may be fluidically isolated or hydraulicallyindependent from one fluids in other tips and from other fluids withinthe device. The fluidically isolated or hydraulically independent unitsmay be movable relative to other portions of the device and/or oneanother. The fluidically isolated or hydraulically independent units maybe individually movable. A fluid handling system may comprise one ormore base or support. A base or support may support one or more pipetteor pipette units. A base or support may connect one or more pipettes ofthe fluid handling system to one another.

A sample processing system, which may include a sample processingdevice, may be configured to perform processing steps or actions on asample obtained from a subject. Sample processing may include samplepreparation, including, e.g., sample dilution, division of a sample intoaliquots, extraction, contact with a reagent, filtration, separation,centrifugation, or other preparatory or processing action or step. Asample processing device may be configured to perform one or more samplepreparation action or step on the sample. Optionally, a sample may beprepared for a chemical reaction and/or physical processing step. Asample preparation action or step may include one or more of thefollowing: centrifugation, separation, filtration, dilution, enriching,purification, precipitation, incubation, pipetting, transport,chromatography, cell lysis, cytometry, pulverization, grinding,activation, ultrasonication, micro column processing, processing withmagnetic beads, processing with nanoparticles, or other samplepreparation action or steps. For example, sample preparation may includeone or more step to separate blood into serum and/or particulatefractions, or to separate any other sample into various components.Sample preparation may include one or more step to dilute and/orconcentrate a sample, such as a clinical sample or biological sample,e.g., of blood, urine, sputum, material obtained from a nasal swab, athroat swab, a cheek swab, or other sample, or other clinical orbiological samples. Sample preparation may include adding ananti-coagulant or other ingredients to a sample. Sample preparation mayalso include purification of a sample. In embodiments, all sampleprocessing, preparation, or assay actions or steps are performed by asingle device. In embodiments, all sample processing, preparation, orassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample processing, preparation, or assayactions or steps are performed by a single device, and may be performedwithin a housing of a single device. In embodiments, many sampleprocessing, preparation, or assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

A sample processing system, which may include a sample processingdevice, may be configured to run one or more assay on a sample, and toobtain data from the sample. An assay may include one or more physicalor chemical treatments, and may include running one or more chemical orphysical reactions. A sample processing device may be configured toperform one, two or more assays on a small sample of bodily fluid. Oneor more chemical reaction may take place on a sample having a volume, asdescribed elsewhere herein. For example one or more chemical reactionmay take place in a pill having less than femtoliter volumes. In aninstance, the sample collection unit is configured to receive a volumeof the bodily fluid sample equivalent to a single drop or less of bloodor interstitial fluid. In embodiments, the volume of a sample may be asmall volume, where a small volume may be a volume that is less thanabout 1000 μL, or less than about 500 μL, or less than about 250 μL, orless than about 150 μL, or less than about 100 μL, or less than about 75μL, or less than about 50 μL, or less than about 40 μL, or less thanabout 20 μL, or less than about 10 μL, or other small volume. Inembodiments, all sample assay actions or steps are performed on a singlesample. In embodiments, all sample assay actions or steps are performedby a single device. In embodiments, all sample assay actions or stepsare performed within a housing of a single device. In embodiments, mostsample assay actions or steps are performed by a single device, and maybe performed within a housing of a single device. In embodiments, manysample assay actions or steps are performed by a single device, and maybe performed within a housing of a single device. In embodiments, sampleprocessing, preparation, or assay actions or steps may be performed bymore than one device.

A sample processing system, which may include a sample processingdevice, may be configured to perform a plurality of assays on a sample.For example, a sample processing device may be configured to detect, orto identify, or to measure pathogen-identifying material in a sample. Inembodiments, a sample processing device may be configured to perform aplurality of assays on a single sample. In embodiments, a sampleprocessing device may be configured to perform a plurality of assays ona single sample, where the sample is a small sample. For example, asmall sample may have a sample volume that is a small volume of lessthan about 1000 μL, or less than about 500 μL, or less than about 250μL, or less than about 150 μL, or less than about 100 μL, or less thanabout 75 μL, or less than about 50 μL, or less than about 40 μL, or lessthan about 20 μL, or less than about 10 μL, or other small volume. Asample processing device may be capable of performing multiplexed assayson a single sample. A plurality of assays may be run simultaneously; maybe run sequentially; or some assays may be run simultaneously whileothers are run sequentially. One or more control assays and/orcalibrators (e.g., including a configuration with a control of acalibrator for the assay/tests) can also be incorporated into thedevice; control assays and assay on calibrators may be performedsimultaneously with assays performed on a sample, or may be performedbefore or after assays performed on a sample, or any combinationthereof. In embodiments, all sample assay actions or steps are performedby a single device. In embodiments, all of a plurality of assay actionsor steps are performed within a housing of a single device. Inembodiments, most sample assay actions or steps, of a plurality ofassays, are performed by a single device, and may be performed within ahousing of a single device. In embodiments, many sample assay actions orsteps, of a plurality of assays, are performed by a single device, andmay be performed within a housing of a single device. In embodiments,sample processing, preparation, or assay actions or steps may beperformed by more than one device.

In embodiments, all of a plurality of assays may be performed in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about two hours, or less than aboutone hour, or less than about 40 minutes, or less than about 30 minutes,or less than about 25 minutes, or less than about 20 minutes, or lessthan about 15 minutes, or less than about 10 minutes, or less than about5 minutes, or less than about 4 minutes, or less than about 3 minutes,or less than about 2 minutes, or less than about 1 minute, or othershort time period.

In embodiments of the methods, devices, and systems disclosed herein,systems may include one or more assay stations, where, for example, asample and a reagent may be mixed. In embodiments of the methods,devices, and systems disclosed herein, systems may include one or moredetection stations, where, for example, a sample, or a label attached toa target in a sample, or other signal indicative of the presence of atarget analyte in a sample, may be detected. In embodiments of themethods, devices, and systems disclosed herein, one or more assaystations may also serve as a detection station. For example, where anassay reacts a reagent or reagents with an analyte within a vessel, thevessel serves as an assay station; where the vessel is transparent to asignal produced by the reaction, and where the vessel is adjacent adetector effective that the signal (and thus the presence orconcentration of the analyte) may be detected, the vessel also serves asa detection station. For example, some nucleic acid analysis methods andsystems provide a heating block (or other temperature-controllingelement) and a detector at, or near to, a position in which a vesselcontaining sample and reagents is disposed during a reaction, and duringdetection of the results of the reaction. In such an embodiment, avessel is not moved from the location of the reaction to a differentlocation for detection of the results of the reaction, so the vessel(and the location and accompanying devices and operative elements)serves as both an assay station and a detection station. In furtherembodiments, a vessel is moved from a location where a reaction occurs,or a sample (or sample-containing) solution is moved from the assayvessel, to a different location or vessel where detection occurs. Insuch an embodiment, where a vessel (or solution) is moved from thelocation of the reaction to a different location for detection of theresults of the reaction, the vessel (and the location and accompanyingassay devices and operative elements) serves as an assay station, and adifferent location (and devices or elements) serves as a detectionstation.

Systems for detecting the presence of one or more of a plurality ofmarkers indicative of an infectious disease in a small-volume clinicalsample include, for example, a) a sample handling system; b) a detectionstation comprising an optical sensor; c) a fluidically isolated samplecollection unit configured to retain a clinical sample; d) an assaystation comprising at least a first and a second fluidically isolatedassay unit, wherein the first unit comprises a first reagent and thesecond unit comprises a second reagent; and e) a controller, wherein thecontroller comprises a local memory and is operatively coupled to thesample handling system and the detection station. Such systems may beconfigured to perform assays with one or both of the first and secondassay units; wherein the local memory of the controller comprises aprotocol comprising instructions for: i) directing the sample handlingsystem to transfer a portion of the clinical sample to the first assayunit and to the second assay unit; and ii) directing the sample handlingsystem to transfer the first assay unit and the second unit assay unitto the detection station. In further embodiments, an assay station insuch systems may include at least a first, second, and third fluidicallyisolated assay unit, wherein the first unit comprises a first reagent,the second unit comprises a second reagent, and the third unit comprisesa third reagent. In further embodiments, an assay station in suchsystems may include at least a first, second, third, and fourthfluidically isolated assay unit, wherein the first unit comprises afirst reagent, the second unit comprises a second reagent, the thirdunit comprises a third reagent, and the fourth unit comprises a fourthreagent. It will be understood that embodiments of such systems mayinclude more than four assay units; or other numbers of assay units.

In embodiments, assays are performed with any one or more of the first,second, and third assay units; or with any one or more of the first,second, third, and fourth assay units, wherein the local memory of thecontroller comprises a protocol comprising instructions for: i)directing the sample handling system to transfer a portion of theclinical sample to the first assay unit, the second assay unit, and,where applicable, the third assay unit and/or fourth assay units; andii) directing the sample handling system to transfer the first assayunit, the second assay unit, and, where appropriate, the third assayunit and/or the fourth assay unit to the detection station.

Further systems for detecting the presence of one or more of a pluralityof markers indicative of an infectious disease in a small-volumeclinical sample include a) a sample handling system; b) a detectionstation comprising an optical sensor; c) a fluid handling systemconfigured to transport fluids between components of said system,wherein said transport of fluids comprises transport of isolatedaliquots of fluid; d) a fluidically isolated sample collection unitconfigured to retain a clinical sample; e) an assay station comprisingat least a first, second, and third fluidically isolated assay unit,wherein the first unit comprises a first reagent, the second unitcomprises a second reagent, and the third unit comprises a thirdreagent; and f) a controller, wherein the controller comprises a localmemory and is operatively coupled to the sample handling system and thedetection station. Such systems may be configured to perform assays withany one or more of the first, second, and third assay units; wherein thelocal memory of the controller comprises a protocol comprisinginstructions for: i) directing the sample handling system to transfer aportion of the clinical sample to the first assay unit, the second assayunit and the third assay unit; and ii) directing the sample handlingsystem to transfer the first assay unit, the second assay unit, and thethird assay unit to the detection station. It will be understood thatembodiments of such systems may include only two assay units; or mayinclude four assay units; or other numbers of assay units.

Clinical sample processing systems for use in performing assays asdisclosed herein may include a) a sample handling system; b) a detectionstation comprising an optical sensor; c) a fluidically isolated samplecollection unit configured to retain a clinical sample; d) an assaystation comprising at least a first, second, and third fluidicallyisolated assay unit, wherein the first unit comprises an antibody, thesecond unit comprises an oligonucleotide, and the third unit comprises achromogen or a dye or other label; and e) a controller, wherein thecontroller is operatively coupled to the sample handling system, whereinthe sample handling system is configured to transfer a portion of theclinical sample from the sample collection unit to each of the firstassay unit, the second assay unit, and the third assay unit, and thedevice is configured to perform an immunoassay, a nucleic acid assay,and a general chemistry assay comprising a chromogen.

Such systems may be point-of service (POS) systems. These systems may becontained within a housing. A system located at a POS location may beconfigured for use in analyzing a sample at the POS location. Thesesystems may be POS systems configured to perform a plurality of assayson a single small volume sample, or on aliquots thereof.

Example 1

Testing for and Detecting Nucleic Acid Markers of Disease andDisease-Causing Agents

Disease-causing agents such as viruses, bacteria, yeast, fungi, andother micro-organisms have identifying nucleic acids and proteins, amongother identifying characteristics which may serve as markers. Markersindicative of a disease, or of a disease-causing agent, such as arespiratory disease, or a form of influenza, or a sexually transmitteddisease, or other disease, include nucleic acid markers.

As shown in the figures, markers for many diseases may be tested for,and may be detected, using nucleic acid assays as disclosed herein. Allof the disease-causing agents tested for in the tests shown in FIG. 1Awere detected within 40 minutes, with most of the agents detected withinabout 30 minutes. Detection times for copy numbers of 100 copies permicroliter (c/μL) were shorter than for lower copy numbers (10 c/μL).The detection time for samples having 100 copies per μL are shown inFIG. 1B; most were near or less than 20 minutes, with many detectiontimes of about 15 minutes or less.

FIG. 1A provides a graphic summary of the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for a range of markers and for twodifferent concentration ranges of the markers (10 c/μl and 100 c/μl,where “c/μl” means copies per microliter (μL)). The 10 c/μl results areshown to the left of the 100 c/μl results for each disease type(influenza (Flu), respiratory, and sexually transmitted disease (STD))shown in the figure. The times are labeled “LOD” (“length of delay”).The vertical axis is shown in units of relative fluorescence (relativefluorescence units, RFU), in thousands.

FIG. 1B provides a bar chart showing the durations of time from theinitiation of nucleic acid assay until detection of the presence of atarget nucleic acid in a sample for the indicated markers for variousdiseases (at 100 c/μl).

Further information regarding detection time for various diseases,grouped by general location of the infection, or type of disease, orsamples by which the diseases may be detected are presented in FIGS. 1Cthrough 1F (all at 100 c/μl). FIG. 1C shows the durations of time fromthe initiation of nucleic acid assay until detection for severalinfluenza strains and identifying targets. FIG. 1D shows the durationsof time from the initiation of nucleic acid assay until detection forseveral respiratory diseases. FIG. 1E shows the durations of time fromthe initiation of nucleic acid assay until detection for severalsexually transmitted diseases. FIG. 1F shows the durations of time fromthe initiation of nucleic acid assay until detection for severaldiseases that can be detected in blood.

Complementary information is presented in Table 2A indicating thenumbers of copies per μl that are detectable with these assays forseveral diseases.

TABLE 2A  1 Trypanosoma cruzi <0.01 c/uL  2 Plasmodium <0.1 c/uL  3Bordetella pertussis <4 c/uL  4 Influenza B <10 c/uL  5 Influenza H3N2<10 c/uL  6 Influenza H1N1 seasonal <10 c/uL  7 Influenza H1N1 novel <10c/uL  8 H7N9 flu-HA gene <10 c/uL  9 H7N9 flu-NA gene <10 c/uL 10 HumanRNaseP <10 c/uL 11 StrepA <10 c/uL 12 Staph. aureus <10 c/uL 13 MRSA <10c/uL 14 Adenovirus B <10 c/uL 15 Adenovirus C <10 c/uL 16 Adenovirus E<10 c/uL 17 Klebsiella pneumoniae KPC <10 c/uL 18 Bocavirus type 2, 4<10 c/uL 19 Coronavirus 229E <10 c/uL 20 Coronavirus NL63 <10 c/uL 21Streptococcus pneumoniae <10 c/uL 22 Bordetella parapertussis <10 c/uL23 Haemophilus parainfluenzae <10 c/uL 24 Enterobacter aerogenes <10c/uL 25 Moraxella catarrhalis <10 c/uL 26 StrepB <10 c/uL 27 HSV <10c/uL 28 Treponema pallidum <10 c/uL 29 Hepatitis B-Assay #1 <10 c/uL 30HIV-1 group M-Assay #1 <10 c/uL 31 HIV-1 group M-Assay #2 <10 c/uL 32HIV-2 GroupA <10 c/uL 33 Dengue Virus type 1 <10 c/uL 34 Dengue Virustype 2 <10 c/uL 35 Dengue Virus type 3 <10 c/uL 36 Dengue Virus type 4<10 c/uL 37 Epstein-Barr Virus <10 c/uL 38 Influenza A <100 c/uL 39 H5N1<100 c/uL 40 MTB <100 c/uL 41 Bocavirus type 1, 3 <100 c/uL 42Klebsiella pneumoniae phoE <100 c/uL 43 Coronavirus HKU1 <100 c/uL 44Coronavirus MERS <100 c/uL 45 Coronavirus OC43 <100 c/uL 46Parainfluenza Virus 1 <100 c/uL 47 Parainfluenza Virus 2 <100 c/uL 48Parainfluenza Virus 3 <100 c/uL 49 Metapneumo Virus (hMPV) A1 <100 c/uL50 Haemophilus influenzae <100 c/uL 51 HepDelta-Assay #1 <100 c/uL 52HepDelta-Assay #2 <100 c/uL 53 HIV-2 GroupB <100 c/uL 54 WNV-2 <100 c/uL

In the above Tables and elsewhere herein, “NA” indicates neuraminidase;“HA” indicates hemagglutinin; “Klebsiella pneumonia KPC” indicatesKlebsiella pneumonia carbapenemase; the “phoE” of “Klebsiella pneumoniaphoE” indicates phosphate transport porin; “MRSA” indicatesMethicillin-resistant Staphylococcus aureus; “Metapneumo Virus (hMPV)”indicates human Metapneumo Virus; “HepDelta” indicates Hepatitis Delta;“WNV” indicates West Nile Virus; “Pan Inf A” and “Pan Inf B” indicateassays generic for all strains of the indicated influenza; and “HAI”indicates hospital acquired infection.

The average detection time for these 54 diseases (at 100 c/μl or less)was less than 23 minutes (22.77 minute average). A smaller subset of 35diseases measured at 10 c/μl or less had an average detection time ofless than 30 minutes (29.11 minute average). These assays, includingassays for these diseases, are suitable for validation for use inClinical Laboratories Improvement Act (CLIA) laboratories. For example,such assays for several forms of influenza (pandemic influenza A,pandemic influenza B, H1N1-Novel, H1N1-Seasonal, and H3N2 influenza)have been performed through CLIA Validation.

These results were obtained by nucleic acid assays as described belowand in U.S. Patent Application 61/800,606, filed Mar. 15, 2013. Forexample, the following results demonstrate testing for, and detectionof, nucleic acid markers indicative of a variety of infectious diseasesin a short period of time. As shown in the figures, many markers may betested for, and may be detected. FIG. 2 shows results for detection ofmarkers for influenza A (seasonal H1N1 strain). FIG. 3 shows results fordetection of markers for influenza A (novel H1N1 strain). FIG. 4 showsresults for detection of markers for influenza A (H3N2 strain). FIG. 5shows results for detection of markers for influenza A (H7N9 strain).FIG. 6 shows results for detection of markers for influenza A (H5N1strain). FIG. 7 shows results for detection of markers for influenza B.FIG. 8 shows results for detection of markers for influenza MatrixProtein. FIG. 9 shows results for markers for tuberculosis (Myobacteriumtuberculosis). FIG. 10 shows results for markers for staphylocccus(Staphylococcus aureus). FIG. 11 shows results for markers forMethicilin-Resistant Staphylococcus aureus (MRSA). FIG. 12 shows resultsfor markers for streptocccus (Streptococcus Group A). FIG. 13 showsresults for markers for Bordetella pertussis. FIG. 14 shows results formarkers for adenovirus B. FIG. 15 shows results for markers foradenovirus C. FIG. 16 shows results for markers for adenovirus E. FIG.17 shows results for markers for Herpes Simplex Virus (HSV). FIG. 18shows results for markers for Treponema pallidum.

Samples obtained from subjects, including small samples from subjects,may be tested for other diseases in addition to the diseases listed inthe figures and in Table 2A. For example, some other diseases which maybe tested for by these methods are listed in Table 2B. The columnlabeled “Panel” indicates the type of disease (where HAI indicatesHospital Acquired Infection, and STD indicates sexually transmitteddisease).

TABLE 2B # Assay Panel  1 Acinetobacter baumannii HAI  2 Bordetellaparapertussis Respiratory  3 Chlamydophila pneumoniae Respiratory  4 RSVA Respiratory  5 Enterobacter aerogenes HAI  6 Hepatitis C STD  7Enterobacter cloacae HAI  8 H. influenzae blaTEM HAI  9 Legionellapneumophila HAI 10 Serratia marcescens HAI 11 Metapneumovirus BRespiratory 12 Pseudomonas aeruginosa HAI 13 Parainfluenza 4aRespiratory 14 Parainfluenza 4b Respiratory 15 West Nile Virus 1Respiratory 16 Penicillin-resistant S. pneumo Respiratory 17 HIV-1 groupO STD 18 H. influenzae blaROB HAI 19 RSV B Respiratory 20 Rhinovirus ARespiratory 21 Rhinovirus B Respiratory 22 Rhinovirus C Respiratory

The systems, methods, and devices disclosed herein may be used to testfor, and to detect, the presence of markers indicative of one or more ofthe infectious agents listed above; such testing, and such detecting,may be performed on a single clinical sample, or on a plurality ofaliquots of a single clinical sample. Such a single clinical sample maybe a single small-volume clinical sample. Such testing, and detecting,may be performed at a POS location; the systems, devices and methods maybe POS systems, device, and methods. For example, the clinical samplemay be collected at the POS location, and may be analyzed in a device atthe POS location. As shown in the results illustrated in the Figures,the analysis of the small-volume clinical sample may be completed in ashort period of time.

Thus, the following are some of the disease-causing agents may be testedfor, and may be detected, according to the methods, and by the systemsand devices, as disclosed herein.

TABLE 3 Disease-Causing Agents and Markers Therefor Influenza A Matrixprotein Influenza H3N2 Influenza H1N1 seasonal Influenza H1N1 novelInfluenza B Streptococcus pyogenes (A) Mycobacterium TuberculosisStaphylococcus aureus (MR) Staphylococcus aureus (RS) Bordetellapertussis (whooping cough) Streptococcus agalactiae (B) Influenza H5N1Influenza H7N9 Adenovirus B Adenovirus C Adenovirus E Hepatitis bHepatitis c Hepatitis delta Treponema pallidum HSV-1, HSV-2 HIV-1 HIV-2Human RNaseP (sample prep control) Dengue 1 Dengue 2 Dengue 3 Dengue 4Malaria West Nile Virus Trypanosoma cruzi (Chagas) Klebsiella pneumoniae(Enterobacteriaceae spp) Klebsiella pneumoniae carbapenemase (KPC)Epstein Barr Virus (mono) Rhinovirus Parainfluenza virus (1)Parainfluenza virus (2) Parainfluenza virus (3) Parainfluenza virus (4a)Parainfluenza virus (4b) Respiratory syncytial virus (RSV) A Respiratorysyncytial virus (RSV) B Coronavirus 229E Coronavirus HKU1 CoronavirusOC43 Coronavirus NL63 Novel Coronavirus Bocavirus human metapneumovirus(HMPV) Streptococcus pneumoniae (penic R) Streptococcus pneumoniae (S)Mycoplasma pneumoniae Chlamydia pneumoniae Bordetella parpertussisHaemophilus influenzae (ampic R) Haemophilus influenzae (ampic S)Moraxella catarrhalis Pseudomonas spp (aeruginosa) Haemophilusparainfluenzae Enterobacter cloacae (Enterobacteriaceae spp)Enterobacter aerogenes (Enterobacteriaceae spp) Serratia marcescens(Enterobacteriaceae spp) Acinetobacter baumanii Legionella sppEscherichia coli Candida Chlamydia trachomatis HPV Neisseria gonorrhoeaeTrichomonas (vagin)

The disease-causing agents listed in Table 3 may be tested for, and maybe detected, by the methods, and using the systems and devices disclosedherein. For example, markers for the disease-causing agents listed inTable 3 may be tested for, and may be detected, by the methods, andusing the systems and devices disclosed herein. Such markers mayinclude, for example, nucleic acid markers. In addition, such markersmay include saccharide markers, or other markers, such as, e.g., proteinmarkers. Methods of testing for, and of detecting, protein markers arediscussed in the following example.

Example 2

Detection of Influenza Virus from 2 μL of Prepared Sample

Detection of nucleic acid from 2 μL of sample taken from cultured cellsinfected with seasonal influenza virus (H1N1) is shown in FIGS. 19A(sample) and 19B (control). Nucleic acid obtained from the cell cultureswas prepared using the Chemagic magnetic separator module I with DWP 24XL adapters and reagents from the Chemagic Viral DNA/RNA Kit (No.CMG-1089; No. CMG-1082 is similar) from Chemagen (PerkinElmer chemagenTechnologie Gmbh, Baesweller, Germany)). This method uses magnetic beadseparation to isolate RNA and DNA from a sample. Chemagen reagents anddisposables were used in preparing the samples.

H1N1 influenza RNA was obtained from cultured infected MDCK cells.Briefly, cell culture samples were prepared by dispensing approximately1 mL of sample solution into a well containing lysis buffer, poly(A) RNAreagent, and proteinase K solutions with gentle mixing. The wells werecovered and heated at 55° C. for ten minutes. Following this ten minuteincubation, binding buffer was added to the wells containing the lysedsample solution. This mixed solution was then processed by the Chemagicmagnetic separator module I. Nucleic acids were released by vortexing(rotation of probes) in the buffer and then bound to the magnetic beads,which were immobilized by a magnet during wash steps. The nucleic acidsfreed in the sample bound to the beads and were retained during washsteps; following the wash steps, the nucleic acids were eluted intoelution buffer (10 mM TRIS-HCl, pH 8.0).

Following this sample preparation, the prepared sample was placed in acontainer held in a cartridge, and the cartridge was loaded on anautomatic sample analysis device (such cartridges, devices, and theiruses are described, for example, in U.S. Pat. No. 8,088,593; U.S. Pat.No. 8,380,541; U.S. patent application Ser. No. 13/769,798, filed Feb.18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb. 18,2013; U.S. patent application Ser. No. 13/769,820, filed Feb. 18, 2013;PCT/US2012/57155, filed Sep. 25, 2012; U.S. patent application Ser. No.13/244,949, filed Sep. 26, 2011; U.S. Application Ser. No. 61/800,606,filed Mar. 15, 2013; U.S. Application Ser. No. 61/766,095, filed Feb.18, 2013; and U.S. Application Ser. No. 61/673,245, filed Jul. 18, 2012;U.S. Patent Application 61/805,923, filed Mar. 27, 2013, herebyincorporated by reference in their entireties).

A 2 μL aliquot of the prepared sample solution was placed in a vesselcontaining 20 μL of MasterMix (containing buffer, betaine, dNTPs,forward (RLX1222) and reverse (RLX1223) probes, Syto 59 Red dye), andmixed with 3 μL of enzyme preparation (containing B. stearothermophilusDNA polymerase (Bst), Avian Myeloblastosis Virus Reverse Transcriptase(AmvRT), NEB4 buffer (New England Biolabs Cat. No. B7004S), and water)in a reaction vessel in the automatic sample analysis device. Primersspecific for H1N1 influenza virus were included in the mixture in thereaction vessel. The combination of sample, MasterMix, template, andenzyme preparation was incubated at 56° C. in the reaction vesselaccording to the methods discussed above, and fluorescence was measuredevery minute for 30 minutes (fluorescence was from SYTO 59 dye). Thefluorescence was read as relative fluorescence.

FIG. 19A shows amplification over time, the rise in relativefluorescence at about 15 to 20 minutes indicating the presence of anInfluenza H1N1 seasonal marker. The horizontal axis is in “minutes; thevertical axis is shown in units of relative fluorescence (relativefluorescence units, RFU).

FIG. 19B shows amplification of “no template control” (no added copiesof the target marker; NTC). Note that most experiments showed noamplification; the three runs that show late increases in relativefluorescence did so at about 25 minutes or later. The horizontal axis isin “minutes; the vertical axis is shown in units of relativefluorescence (relative fluorescence units, RFU).

The results from FIGS. 19A and 19B show that viral nucleic acid can bedetected from small volume samples (e.g., 2 μL of sample) within a shortamount of time (e.g., about 15 to 20 minutes or less).

Example 3

Detection of Influenza Virus Proteins by ELISA

Detection of proteins indicative of Influenza A infection and proteinsindicative of Influenza B infection was accomplished using devices andsystems as described, for example, in U.S. Pat. No. 8,088,593; U.S. Pat.No. 8,380,541; U.S. patent application Ser. No. 13/769,798, filed Feb.18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb. 18,2013; U.S. patent application Ser. No. 13/769,820, filed Feb. 18, 2013;PCT/US2012/57155, filed Sep. 25, 2012; U.S. patent application Ser. No.13/244,949, filed Sep. 26, 2011; U.S. Application Ser. No. 61/800,606,filed Mar. 15, 2013; U.S. Application Ser. No. 61/766,095, filed Feb.18, 2013; and U.S. Application Ser. No. 61/673,245, filed Sep. 26, 2011;U.S. Patent Application 61/805,923, filed Mar. 27, 2013 (references thatwere previously listed and incorporated by reference in their entiretiesin text above). Unless otherwise stated below (e.g., with regard toresults obtained with commercial systems for comparison) such devicesand systems were used to obtain the data presented below.

Assay Design and Purpose:

The assays for Influenza A and Influenza B were designed to providequalitative detection of Influenza A or Influenza B nucleoproteinantigens in a sample obtained with a nasal swab. These assays are usefulin the diagnosis of Influenza A viral infections or Influenza B viralinfections in a subject from whom the sample was obtained. The assay wasa sandwich assay, in which anti-Influenza A or B antibodies wereimmobilized on a substrate (the interior of a translucent or transparentpipette tip), and sample, alkaline phosphatase (ALP)-conjugatedanti-Influenza A or B antibody, and ALP substrate added to producechemiluminescence proportional to the amount of Influenza antigen in thesample. The assay results were compared to those of a commercial test(the Remel X/pect Influenza A & B; Remel Products, Lenexa, Kans., USA, adivision of Thermo Fisher Scientific, Inc.).

Materials and Methods:

The interior of a custom polymer pipette tip served as the surface forthis sandwich ELISA assay. The pipette tips were typically made frompolystyrene or polypropylene, although other polymers or plasticmaterials are also suitable. The pipette tip interiors were coated withavidin. The capture surface for the sandwich ELISA was prepared bycoating biotin-labeled anti-Influenza A antibody or biotin-labeledanti-Influenza B antibody onto the avidin-coated interior surfaces ofthe pipettes.

Capture and detection antibodies were obtained from United StatesBiological Corporation (Salem, Mass., USA) or SouthernBiotech(SouthernBiotechnology Associates, Inc., Birmingham, Ala., USA); captureantibodies were conjugated with biotin using a biotin labeling kit, anddetection antibodies were conjugated with ALP using a ALP labeling kit,both from Dojindo Molecular Technologies, Inc. (Rockville, Md., USA).Buffers were obtained from Sigma Aldrich Corporation (St. Louis, Mo.,USA).

Samples were obtained from the nasal passages of subjects using nasalswabs. Nasal swabs containing sample material were then subjected to anextraction process. ALP-labeled anti-influenza A or ALP-labeledanti-influenza B antibodies were then mixed with the extracted samplematerial. This mixture was then incubated with the capture surface for 5minutes. After the incubation, the capture surface was washed and ALPsubstrate was incubated on the surface for 5 minutes; the resultingchemiluminescence intensity was then read, with results reported inRelative Light Units (RLU).

Buffers:

TRIS-buffered Saline consisted of 138 mM NaCl, 2.7 mM KCl, and 0.05 Mtris(hydroxymethyl)aminomethane (TRIS), pH 8.

The extraction buffer was 0.5% Tween 20, 0.1% sodium azide in 20 mMsodium phosphate buffer (pH 7.6).

The blocking buffer was 3% BSA blocking buffer, consisting ofTRIS-buffered Saline, 3% BSA, 0.05% NaN₃, at pH 8.

The alkaline phosphatase (AP) stabilizer was prepared by adding 0.1 mMzinc chloride and 5 mM magnesium chloride to the 3% BSA blocking buffer.

The wash buffer was TRIS-buffered Saline, 0.05% Tween 20, 0.05% NaN₃, atpH 8.

Influenza A and Influenza B Antibody Screen:

Various permutations of pairs of Influenza A or Influenza B antibodies,consisting of paired capture antibodies (CAbs) and detection antibodies(DAbs), were tested on microtitre plates in order to identify thebest-performing pairs. A volume of 50 μl of sample was added to 400 μlof extraction buffer for these experiments. The conditions included 5μg/mL of CAb and 100 ng/mL (final concentration) of DAb in blockingbuffer. Positive and negative controls were from kits obtained fromeither Microbix Biosystems, Inc. (Mississauga, Ontario, Canada) or theVirusys Corporation (Taneytown, Md., USA). The best pairs from themicrotitre plate screening experiments were then evaluated on thedevices and systems disclosed herein, as discussed in the followingparagraphs.

Capture Surface Titration:

The capture surface was titrated at the following concentrations: 10μg/ml, 5 μg/ml, and 1 μg/ml. Controls from the Virusys kit and Microbixkit were used for this screening. The background control was a blockingbuffer blank with no added sample. The DAb was maintained at aconcentration of 100 ng/ml (final concentration in blocking buffer). Theoptimal CAb concentration was determined to be 5 μg/ml for bothInfluenza A and Influenza B.

Alkaline Phosphatase Stabilizer:

Two alkaline phosphatase stabilizers were tested for use as DAbdiluents. In these experiments, 50 μl of sample was added to 500 μl ofextraction buffer. The CAb concentration was 5 μg/ml while the DAbconcentration was maintained at 100 ng/ml (final concentration after theprotocol run). Both the custom AP stabilizer solution (ingredientslisted above) and the commercial Stabilzyme® AP conjugate stabilizer(SurModics, Inc., Eden Prairie, Minn., USA) worked well. The custom APstabilizer was used in subsequent experiments.

Detection Antibody Titration:

The AP-conjugated DAbs were titrated in the AP stabilizer solution. Thebest modulation between the positive and negative controls was observedat 50 ng/ml final concentration. The Influenza A positive controls wereobtained from Microbix Biosystems, Inc. and ZeptoMetrix Corporation(Buffalo, N.Y., USA), and the Influenza B positive controls wereobtained from Microbix Biosystems, Inc. and Virusys Corporation.

Specificity Tests—Influenza A:

Specificity and cross reactivity studies were performed in extractionbuffer using the sample processing and analysis devices and systems asdisclosed herein. Controls for testing for potential cross-reactantswere obtained from Microbix Biosystems, Inc. The potentialcross-reactants tested were Respiratory Syncytial Virus, Mycoplasmapneumonia, Adenovirus, Parainfluenza A-III, Parainfluenza A-II andParainfluenza A-I. CAb concentration was 5 μg/ml while DAb concentrationwas 100 ng/ml (final concentration after protocol run). Nocross-reactivity was detected in these experiments. Different strains ofInfluenza A and Influenza B were also tested to determine the InfluenzaA specificity in the assay. Both Zeptometrix and Microbix controls(which are prediluted controls) were used for this test. PositiveInfluenza A control swabs from the Remel Xpect Flu kit were also used. Asample volume of 200 μl was mixed with 200 μl of extraction buffer andtested for these prediluted samples. Swabs were processed using 400 μlof extraction buffer for these experiments. In the following tables,relative light unit (RLU) measurements were made in triplicate; “CV %”is calculated by dividing the standard deviation of the threemeasurements by the mean of the three measurements and multiplying by100.

TABLE 4 Specificity Tests-Influenza A Sample Type Sample Mean RLU CV%Microbix POS CTL Influenza A 127832 25.7 Zeptometrix-POS CTL Influenza A24235 10.3 Swab-Remel (FDA) Influenza A 269726 11.2Zeptometrix-Influenza A Brisbane/59/07 202118 10.8 StrainZeptometrix-Influenza A Brisbane/10/07 60655 14.2 StrainZeptometrix-Influenza A Perth/16/2009 36571 14.0 StrainZeptometrix-Influenza A Solomon Islands/03/2006 91428 11.8 StrainVirusys 250 ng/ml of Influenza A 439907 16.3 Mean Positive 156559Microbix Respiratory Syncytial Virus 1744 21.3 Microbix Mycoplasmapneumoniae 1798 23.2 Microbix Adenovirus 1954 24.7 MicrobixParainfluenza A-III 2162 22.0 Microbix Parainfluenza A-II 2110 25.2Microbix Parainfluenza A-I 2108 20.3 Microbix NEG CTL Influenza A/BNegative 2072 28.0 Zeptometrix-Influenza B Lee/40 2042 16.6 StrainZeptometrix-Influenza B Florida/02/2006 2806 16.4 StrainZeptometrix-Influenza B Brisbane/33/2008 2849 15.7 StrainZeptometrix-Influenza B Panama/45/90 2536 26.9 Strain

Specificity Tests—Influenza B:

Specificity and Cross Reactivity Studies were performed in extractionbuffer using the sample processing and analysis devices and systems asdisclosed herein. CAb concentration was 5 μg/ml while DAb concentrationwas 100 ng/ml (final concentration after protocol run). Nocross-reactivity was detected in these experiments.

TABLE 5 Specificity Tests-Influenza B Mean Type Sample RLU CV % MicrobixCTL Influenza B Pos 120127 11.7 Virusys CTL Influenza B Pos 95127 12.1Mean Positive 107627 Negative CTL Negative Influenza B Virusys CTL 196518.3 Cross Reactant Parainfluenza 1 1257 19.3 Cross ReactantParainfluenza 2 1509 19.3 Cross Reactant Parainfluenza 3 1496 5.4 CrossReactant Adenovirus 1169 23.8 Cross Reactant M. Pneumoniae 1979 6.5Cross Reactant Respiratory Syncytial Virus 1313 25.1 Cross ReactantCorynebacterium diptheriae 3081 22.0 Cross Reactant Streptococcuspyrogenes 4388 24.8 Cross Reactant Streptococcus pneumoniae 6902 25.5Cross Reactant CMV 534 11.5 Cross Reactant N. meningitis 3455 14.8 CrossReactant Epstein Barr Virus 1938 8.2 Cross Reactant Measles 1710 23.6Cross Reactant Mumps 2423 10.0 Cross Reactant E. coli 2291 9.2 Mean RLUof cross reactants 2363 Modulation 45.5

Clinical Evaluation of the Influenza A Assay:

The performance of the Influenza A assay using the sample processing andanalysis devices and systems as disclosed herein was compared to theresults obtained with the Remel FDA kit. CAb concentration was 5 μg/mlwhile DAb concentration was maintained at 50 ng/ml (final concentrationafter the protocol was run). For the National Institute for BiologicalStandards and Control (NIBSC, Hertfordshire, UK) influenza strains, 50μl of sample was added to the swab, and the swab was then treated like asample swab. For the Zeptometrix panel controls (prediluted samples),200 μl of sample was mixed with 200 μl of extraction buffer. Sampleswabs were placed in 500 μl of extraction buffer and incubated for 3-5minutes. This extracted sample was then analyzed using the devices andsystems as disclosed herein. Swabs and samples were processed on theRemel FDA kit as directed in the kit instructions.

In the following tables, the “antibody index” (Ab Index) was used todetermine whether or not target influenza antigens were detected in asample. The Ab Index was calculated by dividing the mean RLU by thecutoff value (calculated from the normal samples). The cutoff value wasset equal to the mean (normals) plus 4.5× standard deviation (normals).An Ab Index of less than one indicates that a sample was a normal sample(negative: no target influenza antigens were detected in the sample); anAb Index of greater than one indicates that a sample was a positivesample (positive: target influenza antigens were detected in thesample). The column labeled “Remel FDA” presents the results of theRemel FDA kit on the indicated samples as either positive (+): influenzaA detected, negative (−): influenza A not detected, or “NT”: not tested.

TABLE 6 Clinical Evaluation-Influenza A Ab Remel Type ID# Index FDANormal Clinicals 1 0.02 − 6 0.02 − 7 0.02 − 8 0.02 − 10 0.02 − 11 0.01 −12 0.02 − 13 0.01 − 15 0.02 − 16 0.04 − 17 0.02 − 18 0.02 − 2 0.32 − 30.23 − 4 0.20 − 9 0.05 − 14 0.29 − 19 0.95 − REMEL FDA Swab 2.66 +Zeptometrix CTLS Influenza A POS 1.27 + Zeptometrix Brisbane/10/072.58 + Influenza A Zeptometrix Solomon Islands/03/2006 3.25 + InfluenzaA Zeptometrix New Caledonia/20/99 2.57 + Influenza A ZeptometrixBrisbane/59/07 5.16 + Influenza A NIBSC STANDARDS Panama 45/90 0.06 NTFLU B Strains Influenza Antigen B-Johannesburg 0.06 NT Influenza AntigenB-Guangdong 0.08 NT Influenza Antigen B/Yamanashi/166/98. 0.11 NTInfluenza Antigen B/Malaysia/2506/2004 0.02 NT Influenza AntigenB/Harbin/7/94 0.06 NT B:/Florida 4/2006 0.04 NT NIBSC STANDARDSInfluenza Antigen A/California/7/2009-H1N1 6.88 NT FLU A StrainsInfluenza Antigen A/HongKong/1073/99 (H9N2) 8.56 NT Influenza AntigenA/Cambodia/R0405050/2007 (H5N1) 6.02 NT Influenza AntigenA/mallard/England/727/2006 (H2N3) 5.70 NT Influenza Antigen A/NewYork/107/2003 (H7N2) (NIBRG-109) 7.26 NT Influenza Antigen A/NewYork/55/2004 (H3N2) (NYMC X-157) 6.54 NT

The results of these Influenza A clinical evaluation experiments showedthat all samples with Influenza A antigens tested positive for InfluenzaA, while normal samples and samples with Influenza B antigens did nottest positive for Influenza A; these results were in agreement with theresults obtained with Remel FDA kit.

Clinical Evaluation of the Influenza B Assay:

The performance of the Influenza B assay using the sample processing andanalysis devices and systems as disclosed herein was compared to theRemel FDA kit. CAb concentration was 5 μg/ml while DAb concentration wasmaintained at 50 ng/ml (final concentration after the protocol was run).For the NIBSC influenza strains, 50 μl of sample was added to the swab,and the swab was then treated like a sample swab. For the Zeptometrixpanel controls (prediluted samples), 200 μl of sample was mixed with 200μl of extraction buffer. Swabs were placed in 500 μl of extractionbuffer and incubated for 3-5 minutes. This extracted sample was thenanalyzed using the devices and systems as disclosed herein. Swabs andsamples were processed on the Remel FDA kit as directed in the kitinstructions. As discussed above, the cutoff value was set equal to themean (normals) plus 4.5× standard deviation (normals), and the Ab Indexwas calculated by dividing the mean RLU by the cutoff value. The columnlabeled “Remel FDA” presents the results of the Remel FDA kit on theindicated samples as either positive (+): influenza B detected, ornegative (−): influenza B not detected.

TABLE 7 Clinical Evaluation-Influenza B Ab Remel Type ID# Index FDANormal Clinicals 1 0.02 − 6 0.03 − 7 0.02 − 8 0.02 − 10 0.04 − 11 0.02 −12 0.03 − 13 0.01 − 15 0.02 − 16 0.06 − 17 0.02 − 18 0.03 − 2 0.11 − 30.03 − 4 0.04 − 9 0.05 − 14 0.36 − 19 0.79 − REMEL FDA Pos B Swab10.78 + Zeptometric QC Influenza A POS 0.02 − panel − Influenza ABrisbane/10/07 0.03 − Influenza A Solomon Islands/03/2006 0.01 −Influenza A New Caledonia/20/99 0.02 − Influenza A Brisbane/59/07 0.03 −NIBSC STANDARDS Panama 45/90 14.38 + Influenza Antigen B-Johannesburg2.58 + Influenza Antigen B-Guangdong 21.28 + Influenza AntigenB/Yannanashi/166/98. 6.05 + Influenza Antigen B/Malaysia/2506/20047.53 + Influenza Antigen B/Harbin/7/94 18.21 + B:/Florida 4/2006 19.27 +Influenza Antigen A/California/7/2009-H1N1 0.36 − Influenza AntigenA/HongKong/1073/99 (H9N2) 0.50 − Influenza AntigenA/Cambodia/R0405050/2007 (H5N1) 0.61 − Influenza AntigenA/nnallard/England/727/2006 (H2N3) 0.50 − Influenza Antigen A/NewYork/107/2003 (H7N2) (NIBRG-109) 0.39 − Influenza Antigen A/NewYork/55/2004 (H3N2) (NYMC X-157) 0.12 − Zeptometrix Panel Influenza BLee/40 11.31 + Influenza B Florida/02/2006 2.57 + Influenza BBrisbane/33/2008 12.36 + Influenza B Panama/45/90 5.93 + Influenza BPanama/45/90 4.64 +

The results of these Influenza B clinical evaluation experiments showedthat all samples with Influenza B antigens tested positive for InfluenzaB, while normal samples and samples with Influenza A antigens did nottest positive for Influenza B; these results were in agreement with theresults obtained with Remel FDA kit.

Example 4

Further examples of markers indicative of infectious disease which maybe detected, identified, and analyzed by methods, systems and devicesdisclosed herein are shown in FIGS. 22-32. For example, FIG. 22 listsfurther markers for diseases which are named in the figure, and whichare grouped together as, e.g. nosocomial diseases (listed under theheading “Nosocomial Panel (HAI)”); respiratory diseases (listed underthe heading “Respiratory Panel”); sexually transmitted diseases (listedunder the heading “STD Panel (lesion swabs)” and “STD Panel (blood)”,where the parenthetical expressions “lesion swabs” and “blood” indicatethe source and method of obtaining the sample); infectious diseases(listed under the heading “Infectious Disease Panel”); gastrointestinaldiseases (listed under the heading “Gastrointestinal Panel”); andurinary tract diseases (listed under the heading “Urinary TractInfection Panel”). FIG. 22 also lists controls and additional assays, asindicated by the labels “Controls” and “Additional Assays”.

FIG. 23A shows an influenza panel naming several influenza types whichmay be identified by the methods and devices discussed herein. Thisfigure refers to detection of various types of influenza by nucleic aciddetection methods discussed herein. In this, and subsequent figures, andelsewhere in the application, “LOD” indicates “limit of detection.” Theinfluenza types may be detected at levels indicated in the figure; forexample, influenza A may be detected by the methods, devices and systemsdisclosed herein when present in a sample at less than 100 copies permicroliter (c/uL, where copies refers to copies of the target nucleicacid sequence indicative of influenza A). As indicated in FIG. 23A,other influenza types, such as influenza B and influenza H1N1 (seasonal)can be detected at levels of less than 10 copies per microliter.

FIG. 23B shows inflection times for several influenza types which may beidentified by the methods and devices discussed herein. Target influenzanucleic acids indicative of the named influenza types were tested at 100copies per microliter, and the time (from initiation of the nucleic aciddetection assay) in minutes until detection is displayed (as detected bythe inflection of the RFU output as shown, e.g., in previous figures).

FIG. 24A shows various respiratory disease panels naming respiratorydisease types which may be identified by the methods and devicesdiscussed herein. The limit of detection (LOD) is indicated for eachrespiratory disease in the right-most column of the figure; LODs wereeither 10 copies per microliter (c/uL) or 100 c/uL.

FIG. 24B shows inflection times for upper and lower respiratory tractdisease types which may be identified by the methods and devicesdiscussed herein. Target respiratory disease nucleic acids indicative ofthe named respiratory diseases were tested at 100 copies per microliter,and the time (from initiation of the nucleic acid detection assay) inminutes until detection is displayed (as detected by the inflection ofthe RFU output as shown, e.g., in previous figures).

FIG. 25A shows various hospital acquired infectious diseases (indicatedby the acronym “HAI”) naming diseases which may be identified by themethods and devices discussed herein. The limit of detection (LOD) isindicated for each HAI disease in the right-most column of the figure;all LODs were 10 copies per microliter (c/uL).

FIG. 25B shows inflection times for various hospital acquired infectiousdisease panels naming respiratory disease types which may be identifiedby the methods and devices discussed herein. Target nucleic acidsindicative of the named HAI diseases were tested at 100 copies permicroliter, and the time (from initiation of the nucleic acid detectionassay) in minutes until detection is displayed (as detected by theinflection of the RFU output).

FIG. 26 shows results of a nucleic acid assay as described herein (seealso the descriptions of these methods, e.g., in U.S. PatentApplications 61/800,606; 61/908,027; 62,001,050; and Ser. No.14/214,850) for influenza A matrix protein that is designed to beinclusive for all Influenza A subtypes. The results are specific. Notethat the inflection times for the “no template control” (NTC) as well asfor the influenza B targets were significantly greater than (and readilydistinguishable from) the inflection times for the influenza A targets.

FIG. 27 shows that the nucleic acid assays described herein (see alsothe descriptions of these methods, e.g., in U.S. Patent Applications61/800,606; 61/908,027; 62,001,050; and Ser. No. 14/214,850) arespecific for the target H2N2 influenza type. The results are specific.Note that the inflection times for the H3N2 influenza A targetsA/Aichi/2/68, A/Victoria/3/75, and L881 were significantly shorter than(and readily distinguishable from) the inflection times for the non-H3N2influenzas.

FIG. 28 shows that the nucleic acid assays as described herein (see alsothe descriptions of these methods, e.g., in U.S. Patent Applications61/800,606; 61/908,027; 62,001,050; and Ser. No. 14/214,850) arespecific for the target H1N1 seasonal influenza type. Note that theinflection times for the H1N1 (seasonal) influenza A targets weresignificantly shorter than (and readily distinguishable from) theinflection times for the other influenzas and for the no templatecontrol (NTC).

FIG. 29 shows potential interfering substances for the nucleic acidassays as applied to the sexually transmitted disease (STD) panel, andconcentrations which these interfering substances were tested forinterference with the assays. None of the indicated concentrations ofthe potentially interfering substances interfered with the nucleic acidassays.

FIG. 30 shows potential interfering substances for the nucleic acidassays as applied to the sexually transmitted disease (STD) urine panel,and concentrations which these interfering substances were tested forinterference with the assays. None of the indicated concentrations ofthe potentially interfering substances interfered with the nucleic acidassays.

FIG. 31 shows potential interfering substances for the nucleic acidassays as applied to the blood panel, and concentrations which theseinterfering substances were tested for interference with the assays.None of the indicated concentrations of the potentially interferingsubstances interfered with the nucleic acid assays.

While the above is a complete description of the preferred embodiment asdescribed herein, it is possible to use various alternatives,modifications and equivalents. Therefore, the scope of the presentinvention should be determined not with reference to the abovedescription but should, instead, be determined with reference to theappended claims, along with their full scope of equivalents. Anyfeature, whether preferred or not, may be combined with any otherfeature, whether preferred or not. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means for.” It should be understood that as used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein and throughout theclaims that follow, the meaning of “in” includes “in” and “on” unlessthe context clearly dictates otherwise. Finally, as used in thedescription herein and throughout the claims that follow, the meaningsof “and” and “or” include both the conjunctive and disjunctive and maybe used interchangeably unless the context expressly dictates otherwise.Thus, in contexts where the terms “and” or “or” are used, usage of suchconjunctions do not exclude an “and/or” meaning unless the contextexpressly dictates otherwise.

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as they appear in the US Patent and Trademark Office patent file orrecords, but otherwise reserves all copyright rights whatsoever. Thefollowing notice shall apply: Copyright 2013-2014 Theranos, Inc.

1-216. (canceled)
 217. A method of detecting a disease marker,comprising: a) introducing a cartridge comprising at least two differenttypes of samples into an automatic sample processing device, saidcartridge being configured to hold at least one sample and beingconfigured to hold a swab, wherein said automatic sample processingdevice comprises: i) a sample handling system configured to transport atleast a portion of a sample and being configured to transport anindependently movable assay unit; and ii) an optical detector; b)contacting a sample, or a portion thereof, with a movable assay unit, ora reagent, or both, for the performance of an assay for the detection ofa disease marker, said contacting comprising transporting, with the aidof said sample handling system, at least a portion of said sample, or amovable assay unit, or a reagent, or combinations thereof; c)positioning said sample, or portion thereof, at a location suitable fordetection of an optical signal from the sample or portion thereof bysaid optical detector; and d) detecting the presence of a diseasemarker.
 218. The method of claim 217, comprising performing two or moreassays for the detection of disease markers, and detecting two or moredisease markers in said one or more samples, or in one or more portionsthereof.
 219. The method of claim 217, wherein said one or more samplescomprises a blood sample.
 220. The method of claim 217, wherein said oneor more samples comprises a sample obtained using said swab.
 221. Themethod of claim 220, wherein said sample obtained using said swab wasobtained by swabbing a mouth, a throat, a nasal passage, a vaginal area,or other body cavity of a subject.
 222. The method of claim 217,comprising a first sample and a second sample, wherein said first samplecomprises a sample obtained using said swab, and said second samplecomprises a blood sample.
 223. The method of claim 217, comprisingdetecting the presence of a nucleic acid disease marker and a proteindisease marker.
 224. The method of claim 217, wherein said sample has avolume of less than about 500 microliters.
 225. The method of claim 222,comprising performing two or more assays for the detection of diseasemarkers, and detecting two or more disease markers in said samples, orin one or more portions thereof.
 226. The method of claim 225,comprising detecting the presence of a nucleic acid disease marker and aprotein disease marker.
 227. The method of claim 217, wherein saiddisease marker is selected from a nucleic acid disease marker, a proteindisease marker, a saccharide, a prostaglandin, a cytokine, histamine, asteroid, and a marker for inflammation.
 228. The method of claim 225,wherein said two or more disease markers are selected from a nucleicacid disease marker, a protein disease marker, a saccharide, aprostaglandin, a cytokine, histamine, a steroid, and a marker forinflammation.
 229. The method of claim 217, wherein said disease markeris a marker for inflammation selected from prostaglandins, tumornecrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interferon gamma (IF-γ), bradykinin,complement system molecules, blood-clotting factors, C-reactive protein,erythrocyte sedimentation rate (ESR), white blood cell count, andmorphological changes in blood and other cells. 230-242. (canceled) 243.The method of claim 217, wherein the method is a point-of service methodperformed at a point-of-service location.
 244. The method of claim 222,wherein the method is a point-of service method performed at apoint-of-service location.
 245. The method of claim 217, wherein themethod may be performed in less than about 40 minutes.
 246. The methodof claim 222, wherein the method may be performed in less than about 40minutes. 247-276. (canceled)